Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis
- Elisabetta Catoni†1,
- Marcelo Desimone†1,
- Melanie Hilpert1,
- Daniel Wipf1,
- Reinhard Kunze2,
- Anja Schneider2,
- Ulf-Ingo Flügge2,
- Karin Schumacher1 and
- Wolf B Frommer1Email author
© Catoni et al; licensee BioMed Central Ltd. 2003
Received: 4 November 2002
Accepted: 7 January 2003
Published: 7 January 2003
Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine.
High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH4NO3, arginine or ornithine as sole nitrogen sources.
AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds.
In most organisms, amino acid metabolism is distributed between at least two compartments: cytosol and mitochondria. In addition, plastids contribute significantly to amino acid metabolism of plants . For instance in plants, biosynthesis, conversion and degradation of arginine, ornithine and citrulline are distributed across mitochondria, plastids and cytosol .
In many plants, arginine is stored in seeds as a major nitrogen source, e.g. in Arabidopsis thaliana arginine accounts for 11% of total seed nitrogen . During seed germination, stored arginine is degraded for conversion into transport forms (e.g. glutamine or glutamate). The enzyme arginase, which catalyzes degradation of arginine to ornithine and urea is localized in mitochondria . Thus arginine needs to be imported into mitochondria to allow mobilization. Transport steps across the mitochondrial membranes are also required during biosynthesis. In yeast, ornithine, a precursor of arginine, is synthesized in mitochondria from glutamate, which is imported from the cytosol. Ornithine is transferred to the cytosol before conversion to arginine. Additionally, arginine needs to be imported into mitochondria to allow mitochondrial protein synthesis. Ornithine serves as an intermediate of arginine and proline metabolism, and of the ureide citrulline. In plants, ornithine serves also a precursor for polyamine and alkaloid biosynthesis . Thus also in various other metabolic pathways, transport of arginine, ornithine and also citrulline is required. Although mitochondrial in- and efflux of citrulline was originally described in rat liver and heart , it has not been described in plants.
Thus for metabolite exchange and coordination of all these pathways, multiple carriers must be present on the inner mitochondrial membrane of the different organisms. In the yeast genome, 35 members of the mitochondrial carrier family (MCF) were identified; 12 of them were characterized in terms of substrate specificity . MCF proteins have similar structural features: a relatively low molecular mass of around 30 kDa, a tripartite structure (three repeats of about 100 amino acids), each consisting of two transmembrane-helices separated by a hydrophilic loop. Furthermore, a consensus sequence [P-X-(DE)-X-(LIVAT)-(RK)-X-(LRH)-(LIVMFY)], called the "mitochondrial energy signature", is present in all carriers in one to three copies at the C-terminal end of the first helix of each repeat . Most transporters belonging to this family function as antiporters. A combination of transport studies in mitochondria [8, 9] and reconstitution of carriers expressed in E. coli lead to the identification of the first mammalian mitochondrial carrier gene belonging to the MCF family . Since then, mitochondrial transporter genes encoding various predicted transport systems have been identified in yeast and human genomes [11–14].
The Arabidopsis genome contains at least 48 members of the MCF family, 7 with 3 copies of the mitochondrial energy transfer signature, 21 with two copies and 20 with one copy. Until now, only few plant MCF carriers have been characterized : the adenine nucleotide translocators [15, 16], uncoupling proteins [17, 18] and recently also a dicarboxylate-tricarboxylate carrier named DTC  and a succinate transporter . However, despite their importance none of the postulated plant mitochondrial transporters for amino acids has been identified, such as proline transporters for the compartmentalized proline metabolism , GABA transporters for the GABA shunt , glutamate/aspartate transporters for photorespiratory exchange with peroxisomes or ornithine/arginine carriers for arginine metabolism that were biochemically characterized in isolated soybean mitochondria .
A yeast arg11 mutant unable to synthesize arginine was identified . The corresponding gene was cloned by complementation of the arg 11 mutant, revealing that Arg11p is a member of MCF. On the basis of amino acids content of the mutant, it was suggested that ARG11p would function as an mitochondrial ornithine translocator of yeast . The properties of Ort1p were characterized after expression in E. coli and subsequent reconstitution. The major function of Ort1p/Arg11p is ornithine export from mitochondria. Ort1p/Arg11p mediates electroneutral antiport of ornithine, arginine and lysine, and with lower efficiency also ornithine/H+ antiport . Since mitochondria of an arg11 deletion mutant retained arginine/ornithine carrier activity, a second transporter for basic amino acids named Bac1 was postulated . Sequence homologies to the yeast gene were used to identify the human ORNT1 transporter and to demonstrate that the HHH syndrome (Hyperornithinaemia-Hyperammoniaemia-Homocitrullinuria) is due to defects in the respective gene . In the case of citrullinaemia, a different member of the MCF family was defective , namely the aspartate/glutamate carrier . Yet another amino acid carrier identified by reconstitution in liposomes was the human glutamate transporter . However in yeast, and despite extensive attempts, none of the crucial carriers for amino acids or other metabolites was identified in genetic studies apart from arg11, acr1 and dic1, i.e. mutants have been found for all metabolic steps in many of the amino acid pathways but not the respective mutations in mitochondrial carriers. Since the yeast genome contains a large number of MCF proteins one may hypothesize that transporters have overlapping substrate specificities and yeast may thus be able to compensate loss of single MCF genes.
To date, the most efficient approach to identify plant plasma membrane transporters has been suppression cloning in yeast mutants. Since in most cases mitochondrial transporters do not contain identifiable targeting sequences, it remained open whether yeast complementation may be a suitable tool to identify the plant homologs. Searches in the Arabidopsis genome identified a gene 32% identical to the yeast Ort1p/Arg11p. The Arabidopsis gene AtmBAC2, when expressed in yeast, was able to restore growth of the yeast arg11 mutant on media lacking arginine, indicating that AtmBAC2 catalyzes the export of ornithine from mitochondria to the cytosol. RT-PCR analysis and promoter-GUS fusions were used to study expression and regulation of AtmBAC2. Absence of expression in developing seeds and in cotyledons of seedlings indicated that, in addition to AtmBAC2, another transporter, supposedly AtmBAC1 , is involved in storage and mobilization of arginine.
Results and Discussion
Complementation of the yeast arg11mutant by a plant cDNA
So far, the most efficient methodology to characterize mitochondrial transporters was a candidate gene approach. Candidates were expressed in E. coli , purified proteins were reconstituted in lipid vesicles and transport properties were determined . The approach is difficult and laborious and no standard protocols applicable for all candidates are available . Therefore, an alternative method was developed allowing identification of mitochondrial carriers via suppression of auxo- or bradytrophic phenotypes of yeast mutants. Although there is no evidence for a mitochondrial targeting sequence in the AtmBAC2 sequence, and little is known about signals for import of nuclear encoded proteins in the mitochondria of yeast and plants, the yeast import mechanisms are able to recognize also plant mitochondrial proteins and target them to mitochondria. In this way, the identification of AtmBAC2 and of AtmBAC1  from Arabidopsis as ornithine carriers confirms the validity of yeast complementation for the functional assignment of candidate genes.
Phylogenetic tree of MCF translocators
Expression pattern of AtmBAC2in Arabidopsis
Basic amino acid transporters in the inner mitochondrial membrane are supposed to play roles in arginine metabolism in developing seedlings and during the mobilization of proteins, e.g. during seed germination. Unexpectedly, no expression was found in developing seeds or in young seedlings during germination, suggesting that a different carrier, potentially AtmBAC1, might be responsible for these processes . Proline represents by far the most abundant amino acid in pollen . Thus the expression of AtmBAC2 in pollen may be taken as a hint that arginine can serve as a precursor for proline biosynthesis, since arginase is localized in mitochondria, and thus arginine has to be taken up into mitochondria. Alternatively, the expression pattern may also be relevant for other metabolic pathways since ornithine can also serve as a precursor for polyamine and alkaloid biosynthesis .
Functional expression of a candidate gene under control of a strong promoter allowed identification of a plant mitochondrial transporter for basic amino acids. This carrier seems to have overlapping, but different functions as AtmBAC1 in the plant. The study also pinpoints candidates for the missing amino acid transport systems in plant mitochondria. RNAi studies together with knock-out mutants will enable a detailed physiological characterization of the mitochondrial compartmentation of amino acid biosynthesis. The studies also demonstrate that in addition to the 53 potential amino acid transporters for plasma membrane and tonoplast , plants contain additional carriers for organellar compartmentation.
The yeast strain Y02386 [Matα; his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YOR130c::kanMX4] from Euroscarf (Frankfurt, Germany), carrying a deletion of the yeast gene ARG11, was used for yeast complementation. Selective conditions for Y02386 were established by using yeast nitrogen base without amino acids and ammonium sulfate (Difco, Heidelberg, Germany) supplemented with (mg L-1): 5000 (NH4)2SO4, 20 histidine, 100 leucine, 20 methionine with 2% glucose and eventually 50 uracil in case of untransformed yeast. As negative control Y02386 was transformed with the empty vector pDR195 .
Cloning of the AtmBAC2gene and yeast transformation
AtmBAC2 cDNA was amplified by PCR on an Arabidopsis cDNA library (ecotype Landsberg; ) using primers spanning the predicted ORF from ATG to stop: 5'-agcctcgagatggatttctggccggagtttatg, containing a XhoI restriction site and 5'-ttcggatcctcaatctcctgtgacaatatc, containing a BamHI restriction site. The PCR product was restricted with XhoI and BamHI and ligated into the yeast expression vector pDR195. The resulting plasmid was amplified in E. coli DH5 and transferred into Y02386, according to Gietz et al. . cDNA and protein sequence were deposited in Genbank under the accession number AC011717_10.
Plant material and mRNA expression
Arabidopsis thaliana L. Heynh. ecotype Col-0 was used for plant studies. For organ specificity studies, all plant organs were harvested from plants grown in soil in the greenhouse with the exception of roots, which were obtained from plants cultured on MS agar plates. For expression studies with different nitrogen sources, Arabidopsis seeds were placed on sterile N-free MS-medium supplied with 10 mM nitrogen either as NH4NO3, arginine or ornithine. After 5, 8, 10 and 11 days, plants were harvested and total RNA extracted. For RT-PCR analysis, RNAs were converted to cDNA using Retroscript (Ambion) following the manufacturer's instructions. A 267 bp cDNA fragment was amplified by 25 PCR cycles using specific primers for AtmBAC2 (5'-GGACAAGAAAACCTCAGGACCAT, 5'-GAATAGACACCGCAAGGCTACCT). As a control, a 377 bp cDNA fragment of the constitutively expressed actin 2 gene (AtACT2) was simultaneously amplified by 20 PCR cycles . Identity of the amplicons was confirmed by sequencing.
For promoter-GUS fusions two different lengths (2020 and 1480 bp) of the AtmBAC2 promoter region including the start codon were amplified by PCR on Arabidopsis thaliana Heynh., ecotype Col0 genomic DNA using the primers 5'-CTGACCTCTAGACCAGCTGGTGACAGCATCAG; 5'-CTGACCTCTAGAAAACTTGGGTTTGAACAGCC and 5'-CTGACCACCCGGGACCATACCAAGGATGGGATTGTAC. Forward primers contained a XbaI restriction site, the reverse primer a SmaI site. The PCR product was restricted with SmaI and XbaI and ligated in frame to the GUS gene in binary vector pCB308 .Agrobacterium tumefaciens GV2260 was transformed with the resulting plasmids. Arabidopsis thaliana L. Heynh., ecotype Col0, grown in soil in the greenhouse, was transformed by floral dipping . Transgenic Arabidopsis plants were selected for BASTA resistance (Aventis Crop Science, Frankfurt).
Histochemical analysis of GUS activity
Transformed T2 Arabidopsis plants were grown in soil. At different developmental stages, plants or organs were harvested and stained for GUS activity. Seedlings were grown on solid MS plates. Histochemical assays for β-glucuronidase activity in transgenic plants were performed as described in Martin et al. . Tissues were incubated for ~18 h at 37°C in staining solution containing (mM) 100 sodium phosphate (pH 7), 10 EDTA, 3 K4 [Fe(CN)6], 0.5 K3 [Fe(CN)6], 1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) and 0.2% (v/v) Triton X-100. Slight vacuum was applied to facilitate substrate infiltration. Chlorophyll containing tissues was cleared in 70% EtOH. Photographs were taken on a CCD camera (Visitron).
We would like to thank Rainer Schwacke, Köln, for help with identifying MCF proteins. This work was supported by Bundesministerium für Bildung und Forschung in the framework of "Genomanalyse im biologischen System Pflanze – GABI" and by KWS AG and Südzucker AG.
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