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Regulation of sulfur assimilation in higher plants a sulfate transporter...

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Regulation of sulfur assimilation in higher plants a sulfate transporter...

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     Proc. Natl. Acad. Sci. USA Vol. 94, pp. 11102?C11107, September 1997 Plant Biology

     Regulation of sulfur assimilation in higher plants: A sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis thaliana

     HIDEKI TAKAHASHI*, MAMI YAMAZAKI*?, NORIKO SASAKURA*, AKIKO WATANABE*, THOMAS LEUSTEK?, JANICE DE A LMEIDA ENGLER?, GILBERT ENGLER?ì, MARC VAN MONTAGU?, AND KAZUKI SAITO*?

     *Laboratory of Molecular Biology and Biotechnology, Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan; ?Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, B-9000, Gent, Belgium; ?Center for Agricultural Molecular Biology, Department of Plant Science, Rutgers University, New Brunswick, NJ 08903-0231; and ?ìLaboratoire Associe de ?ä l??Institut National de la Recherche Agronomique (France), Universiteit Gent, B-9000, Gent, Belgium

     Contributed by Marc Van Montagu, July 7, 1997

     ABSTRACT Proton sulfate cotransporters in the plasma membranes are responsible for uptake of the environmental sulfate used in the sulfate assimilation pathway in plants. Here we report the cloning and characterization of an Arabidopsis thaliana gene, AST68, a new member of the sulfate transporter gene family in higher plants. Sequence analysis of cDNA and genomic clones of AST68 revealed that the AST68 gene is composed of 10 exons encoding a 677-aa polypeptide (74.1 kDa) that is able to functionally complement a Saccharomyces cerevisiae mutant lacking a sulfate transporter gene. Southern hybridization and restriction fragment length polymorphism mapping confirmed that AST68 is a single-copy gene that maps to the top arm of chromosome 5. Northern hybridization analysis of sulfate-starved plants indicated that the steady-state mRNA abundance of AST68 increased specifically in roots up to 9-fold by sulfate starvation. In situ hybridization experiments revealed that AST68 transcripts were accumulated in the central cylinder of sulfate-starved roots, but not in the xylem, endodermis, cortex, and epidermis. Among all the structural genes for sulfate assimilation, sulfate transporter (AST68), APS reductase (APR1), and serine acetyltransferase (SAT1) were inducible by sulfate starvation in A. thaliana. The sulfate transporter (AST68) exhibited the most intensive and specific response in roots, indicating that AST68 plays a central role in the regulation of sulfate assimilation in plants.

    In higher plants, sulfur metabolism is initiated by the uptake of sulfate by roots from the environment. Plants assimilate inorganic sulfate into Cys, the first sulfur-containing amino acids, and various sulfur-containing secondary metabolites. Thus, plants serve as nutritional sulfur sources for animals (1). Uptake of sulfate by plants is considered to be the key entry step of the sulfur cycle in the nature. Because the sulfate transporter protein is involved in this initial step, it may play a central role in the regulation of the entire sulfur metabolism pathway by controlling the import of available sulfate. As yet, no detailed molecular biological investigation has been carried out for this important process. Isolation of cDNA clones encoding sulfate transporters has been recently reported for tropical legume Stylosanthes hamata (2), Arabidopsis thaliana (3), and barley (Hordeum vulgare) (accession no. U52867). From our recent studies (3), there were at least three different sulfate transporter homologues in A. thaliana showing different expression patterns. After entry into root cells, sulfate is delivered to various parts of tissues through the vascular

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     1997 by The National Academy of Sciences 0027-8424 97 9411102-6$2.00 0 PNAS is available online at http: www.pnas.org.

     system. The process of ????long-distance translocation???? (4) of sulfate may require several types of transporters responsible for cell-tocell movement of sulfate across the plasma membrane. Loading of sulfate into the vascular tissues in roots and unloading of sulfate into the leaf cells are assumed to be the two essential steps in this process. In the present study, we have shown that these two events are possibly controlled by the same sulfate transporter gene, AST68, in A. thaliana. With respect to the regulation of the entire sulfate assimilation pathway, fluctuation of the extracellular sulfate concentration may act as a signal for the modulation of gene expression. From the earlier physiological experiments on membrane vesicles (5) and cell cultures (6), it is demonstrated that plants can adapt to low sulfate availability by modulating the sulfate transport activity. Because nearly all the genes for the sulfate assimilation enzymes of A. thaliana have been cloned or reported in the expressed sequence tag (EST) database, it is now quite possible to determine which proteins or enzymes in the Cys biosynthesis are regulated. The present study is the first report of a complete analysis of the regulation of the sulfate assimilation pathway in plants. The results show which of the biosynthetic steps are regulated at the level of mRNA expression during

adaptation to sulfate deficiency.

     MATERIALS AND METHODS

     Plant Materials and Yeast and Bacterial Strains. A. thaliana ecotype Columbia was grown on germination medium (GM) agar medium (7) at 22?ãC under 16-h 8-h light and dark cycles. Three-week-old plants were subjected to sulfate starvation for 2 days on the sulfate-deficient GM agar medium in which the sulfate salts were replaced with equivalent amounts of chloride salts. As a control experiment, plants were transferred to the fresh GM agar medium, which was subsequently cultured for 2 days. For complementation studies, S. cerevisiae strain YSD1 (Mat , his3- 1, leu2, trp1?C289, ura3?C52, sul1), a disruption mutant of the yeast sulfate transporter gene SUL1 was provided by M. J. Hawkesford (University of Bristol, U.K.) (8). E. coli strains Y1088 (supE, supF, metB, trpR, hsdR , hsdM , tonA21, strA, lacU169, mcrA, proC::Tn5 pMC9), K802 (galK2, galT22, hsdR2(rk , mk ), lacY1, mcrA , mcrB , metB1, mrr , supE44) and XL1-Blue [endA1, gyrA96, hsdR17, lac , recA1, relA1, supE44, thi-1, (F

     Abbreviations: APS, adenosine 5 -phosphosulfate; CS, cysteine synthase; EST, expressed sequence tag; MSD, membrane spanning domain; OAS, O-acetylserine; PAPS, 3 -phosphoadenosine 5 phosphosulfate; SAT, serine acetyltransferase; GM, germination medium. Data deposition: The sequences reported in this paper have been deposited in the DDBJ EMBL GenBank databases [accession nos. AB003591 (cDNA) and AB003590 (genomic)]. ?To whom reprint requests should be addressed at: Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1?C33, Inage-ku, Chiba 263, Japan. e-mail: ksaito@p.chiba-u.ac.jp.

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     Plant Biology: Takahashi et al. lacqZ M15, proAB, Tn10)] were used for propagation of gt11, EMBL3, and plasmid DNAs, respectively. Isolation of cDNA and Genomic Clones. Approximately 5.0 105 amplified plaques of the gt11 cDNA library of A. thaliana ecotype Columbia (3) were screened with the 32P-labeled cDNA insert of an A. thaliana EST, 142F20T7 (accession no. T76088) (9). Hybridization of the membranes (Hybond N , Amersham) was carried out at 65?ãC in 5 SSPE (0.9M NaCl 0.05 M sodium phosphate, pH 7.7 5 mM EDTA), 0.5% SDS, 5 Denhardt??s solution, and 20 mg liter salmon sperm DNA. Final washing of the membranes was conducted at 65?ãC in 0.1 SSPE and 0.1% SDS (10). For isolation of genomic clones, approximately 1.0 105 amplified plaques of the Arabidopsis EMBL3 genomic library (CLONTECH) were screened with the 32P-labeled cDNA insert of AST68. Hybridization and washing of the membranes (Hybond N , Amersham) were carried out in the same condition as described for the screening of the gt11 cDNA library. DNA Sequencing and Determination of the Transcriptional Start Point. cDNA and genomic fragments of the isolated clones were cloned in the appropriate cloning

    sites of pBluescript II SK( ) (Stratagene). All clones were sequenced on both strands using a series of overlapping exonuclease III digested clones created with the Exo Mung deletion kit (Stratagene). Sequencing was carried out by the dideoxy-chain termination method using Thermo Sequenase (Amersham) and a Shimadzu DNA sequencer model DSQ1000. The primer extention experiment was carried out as described in ref. 11 with some modification. Total RNA (20 g) from A. thaliana leaves was annealed with the synthetic oligonucleotide (10 pmol, 5

    -TGAATATAAAATGTCAGGGA-3 ) prepared at the position of 220 nucleotides upstream of the translational initiation codon. Reverse transcription was carried out by 40 units of Moloney murine leukemia virus reverse transcriptase (United States Biochemical) at 37?ãC in the presence of 0.1 mM of dNTP and 1.85 MBq of [ -32P]dCTP. The extention product was separated in a 5% polyacrylamide sequencing gel, and the signal was detected by the BAS-2000 image analyzer (Fuji). Sequencing products of M13mp18 with the M13-P4 oligonucleotide primer were used as size markers. Complementation of a Yeast Mutant. The coding sequence of the Arabidopsis cDNA clone AST68 was amplified by PCR as a BamHI-ended DNA fragment with a set of synthetic oligonucleotide primers (5 -CT TGGATCCATGA A AGAGAGAGATTCAGAG-3 and 5

    -ATTGGATCCTGGTCCTTTGAAAACTGTTTC-3 ), using Pfu DNA polymerase (Stratagene). The amplified fragment was inserted in the BamHI site of an yeast expression vector, pYES2 (Invitrogen), under control of the GAL1 promoter. The resultant plasmid, pYAT68, was used for the transformation of YSD1 (8) by the electrotransformation method (12). Transformants were replica plated onto synthetic minimal media (13) containing galactose (20 g liter) as a carbon source and 0.1 mM MgSO4 as a sulfur source (8) and incubated at 30?ãC. Southern Hybridization Analysis. Genomic DNA was isolated from the leaves of 3-week-old A. thaliana plants (14). Restriction fragment length polymorphism (RFLP) mapping of AST68 was carried out by the 30 recombinant inbred (RI) lines (15). Genomic DNA (5 g) was digested with restriction enzymes, separated in a 0.7% agarose gel, and transferred to a Hybond N membrane (Amersham). DNA blots were probed with the 32P-labeled full-length cDNA fragment of AST68. Hybridization and washing of the membranes were carried out as described for library screening. Hybridization signals were detected by the BAS-2000 image analyzer (Fuji). The map distance was calculated by C. Lister (John Innes Institute, Norwich, U.K.), based on the RFLP patterns in the RI lines generated by DraI digestion of genomic DNAs. Northern Hybridization Analysis. Total RNA was isolated from the leaves and roots of 3-week-old A. thaliana plants by a phenol SDS method and precipitated by LiCl as described in ref. 10. For RNA blot analyses, 20 g of total RNA was separated

     Proc. Natl. Acad. Sci. USA 94 (1997)

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     under denaturing conditions in a 1.0% agarose gel containing formaldehyde and transferred to Hybond N membranes (Amersham). RNA blots were hybridized with 32P-labeled probe DNAs synthesized from cDNA fragments AST68 (this study), AST56 (3), EST-76E7T7 (accession no. T21459) (9), APS1 (16), ASA1 (17) (identical with APS2) (18), APK1 (19) (identical with ATG1 1) (20), APR1 (21) (identical with PRH-19) (22), SAL1 (23), SIR (24), OAS-TL 5?C8 (25) (identical with AT-CYS-3A) (26), OAS-TL 7?C4 (25), SAT1 (27) (identical with SAT5) (28), SAT-A (29) (identical with Sat-1) (30), and SAT52 (accession no. U30298). The EST clone, 201K3T7 (accession no. H77005) (9), which is identical with SIR (24), was obtained from the Arabidopsis Biological Resource Center of Ohio State University. cDNA fragments of SAL1 (23) and SAT52 were amplified from the cDNA library by PCR with synthetic oligonucleotides prepared according to the reported nucleotide sequence in the database. OAS-TL 5?C8 (25), OAS-TL 7?C4 (25), and SAT-A (29) were provided by R. Hell (Ruhr-Universitat Bochum, Germany). ?? ASA1 (17) was provided by J.-C. Davidian (Ecole Nationale Superieure Agronomique de Montpellier, France). Relative values of mRNA transcripts were calculated based on the hybridization intensities of specific signals on the blot quantified by the BAS-2000 image analyzer (Fuji). To verify equivalent loadings of RNA on blots, membranes were probed with a 32P-labeled rice rDNA (pRR217) (31). Hybridization and washing conditions were the same as described above. In Situ Hybridization. In situ hybridization experiments were carried out on A. thaliana and radish. Thirteen-day-old A. thaliana and 5-day-old radish were transferred to sulfate-deficient medium for 2 days. Radish and A. thaliana seedlings grown in sulfatestarved and control conditions were fixed in 4% formaldehyde, 50% ethanol, and 5% acetic acid for 3 hr at room temperature. Fixed tissues were dehydrated and embedded in paraffin according to standard procedures. Ten-micrometer sections were mounted onto slides coated with 3-aminopropyltriethoxysilane and pretreated for hybridization according to Angerer and Angerer (32). 35S-UTP-labeled sense and antisense mRNA were generated by run-off transcription with T7 and T3 RNA polymerase (Promega). Labeled mRNA probes were hydrolyzed to an average length of 300 nt (32). The hybridization mix contained 35S-labeled mRNA (5 106 cpm per slides), 10 mM Tris HCl (pH8.5), 50% formamide, 0.3M NaCl, 1 mM EDTA, 150 g ml 1 yeast tRNA, 1 Denhardt??s, 10% dextran sulfate, and 70 mM DTT. RNase treatment, washing steps, coating with Kodak NBT2 emulsion, and the development of slides were performed as described in ref. 32. Sections were stained with toluidine blue, dehydrated, and mounted in mounting medium (Depex). Photographs were taken in a Diaplan microscope (Leica) using dark-field optics (Fuji ASA 100 film).

     RESULTS

     Cloning and Functional Identification of a Sulfate Transporter Gene, AST68. An Arabidopsis EST clone, 142F20T7 (accession no. T76088) (9), exhibiting high sequence similarity with the sulfate transporters of the tropical legume S. hamata (2), was used as a probe for isolation of a full-length clone from a gt11 cDNA library. The isolated cDNA clone, AST68, contained a 2.4-kb-length insert that revealed to have an ORF encoding a polypeptide of 677 aa (Fig. 1B). The presence of an in-frame TAG termination codon 39 nucleotides upstream of the translational initiation codon ensured that this ORF encodes the full coding region of the AST68 polypeptide. The sulfate uptake function of the AST68 polypeptide was tested in the yeast sulfate transporter mutant strain YSD1 (8). Expression of AST68 allowed YSD1 to grow on the minimal medium containing 0.1 mM of sulfate as a sole sulfur source only in the presence of galactose (Fig. 2). The strain was unable to grow with glucose (not shown). These data confirmed that AST68 encodes a functional homologue of a yeast sulfate transporter (8).

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     Proc. Natl. Acad. Sci. USA 94 (1997)

     FIG. 1. Structure of the AST68 gene. (A) The partial restriction map of the AST68 genomic region. Solid bars and open bars indicate exons and introns, respectively. The transcriptional starting point (tsp) and the translational initiation (ATG) and termination (TAA) codons are indicated by arrows. Abbreviations of restriction enzymes are as follows: E, EcoRI; Xb, XbaI; Xh, XhoI. (B) Alignment of the deduced amino acid sequences of the plant sulfate transporters: Arabidopsis AST68, Arabidopsis AST56 (3), Stylosanthes hamata SHST1, SHST3 (2), Holdeum vulgare HVST1 (accession no. U52867). Consensus amino acids are indicated by asterisks. Gaps in the sequence inserted to obtain the best alignment are indicated by dashes. Insertion sites of the introns of the AST68 gene are indicated by arrowheads. Solid bars indicate 12 putative MSDs. A conserved basic amino acid residue (Arg-407) between MSDs 9 and 10 is indicated by a solid circle.

     A genomic clone corresponding with the cDNA clone, AST68, was isolated from a EMBL3 library. Several clones were isolated that hybridized with AST68. Sequence analysis of a 5-kb XbaI fragment (Fig. 1 A) from one of these clones revealed that the AST68 gene consists of 10 exons and 9 introns. All of the exon intron junctions had the consensus GT AG splice donor and acceptor sites. Primer extension experiments were carried out with a synthetic oligonucleotide primer prepared according to the sequence of the AST68 genomic clone. The transcriptional start point (tsp) was located 285 nucleotides upstream of the translational initiation codon (Fig. 1 A). The 5 untranslated

    region between the tsp and the 5 end of the cDNA clone was amplified by reverse transcriptase?CPCR (RT-PCR) for the determination of the nucleotide sequence. Predicted Structure of the AST68 Polypeptide. The hydropathy profile of AST68 predicted by the TOPPREDII program (33) indicated the presence of 12 hydrophobic membrane spanning domains (MSD). This structural feature was well conserved in eukaryotic membrane-bound transporter proteins. According to this model, several basic amino acid residues (Lys, Arg) were located on both sides of the membrane. Among them, Arg-407, which is located between MSDs 9 and 10, was the only basic residue identical to the other known eukaryotic sulfate trans-

     porters (Fig. 1B). As previously indicated by Smith et al. (2), this residue may have some functional significance for binding of the sulfate anion on the membrane surface. The calculated model also predicted the putative N-glycosylation site at Asn-255 between MSDs 5 and 6 to be located on the extracellular side. Phylogenic Relationship. Similarities between the amino acid sequence and other eukaryotic sulfate transporters were calculated by the GENETYX program (Software Development, Tokyo) as follows: S. cerevisiae Sul1, 28% (accession no. X82013) (8); N. crassa Cys14, 21% (accession no. M59167) (34); S. hamata Shst1, 49% (accession no. X82255) (2); S. hamata Shst2, 49% (accession no. X82256) (2); S. hamata Shst3, 64% (accession no. X82254) (2); A. thaliana AST56, 63% (accession no. D85416) (3); soybean nodule GmN#70, 54% (accession no. D13505) (35); H. vulgare Hvst1, 51% (accession no. U52867); mouse, 28% (accession no. D42049); human DTD, 28% (accession no. U14528) (36); rat Sat-1, 27% (accession no. L23413) (37); and human DRA, 25% (accession no. L02785) (38). The phylogenic relationship of these amino acid sequences indicated that the two sulfate transporters of A. thaliana, AST56 (3) and AST68 (this study), fall into a group that includes the plant low-affinity transporters. In spite of the structural and functional similarity, AST68 is highly divergent from the yeast sulfate transporter, SUL1 (8).

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     FIG. 2. Complementation analysis of AST68. A wild-type strain, INVSc1, and the sulfate transporter-deficient mutant, YSD1, transformed with vectors pYES2 (control) and pYAT68 (harboring the AST68 coding region), were grown at 30?ãC on the synthetic minimal media containing galactose (20 g liter) as a carbon source and 0.1 mM MgSO4 as a sulfur source as described in ref. 9.

     Chromosomal Location of the AST68 Gene. Southern blot analysis of the AST68 gene was carried out for the A. thaliana genomic DNA digested with several restriction enzymes. Restriction with BamHI, EcoRV, SacI,

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