We thank Dr. J. Giraudat, Gif-Sur-Yvette, France, for the Arabi- dopsis cDNA library, F. Grellet for help with DNA sequence analysis, and Dr. Richard Cooke for ...
Plant Physiol. (1993) 102: 327-328
Plant Gene Register
Nucleotide Sequence of a cDNA Clone Encoding an Arabidopsis thaliana T h ioredoxin h Renata Rivera-Madrid*', Paulo Marinho', Christophe Brugidou, Yvette Chartier, and Yves Meyer
Laboratoire de Physiologie Moléculaire Végétale, Unité de Recherche Associée au Centre National d e Ia Recherche Scientifique 565, avenue de Villeneuve, 66860 Perpignan, France a9
Thioredoxins are small proteins of approximately 12 kD that reduce disulfide bridges of target proteins by the reversible formation of a disulfide bridge between two neighboring Cys residues present in the active site (Try-Cys-Gly-Pro-Cys). Thioredoxins have been found to regulate a variety of biological reactions in prokaryotic and eukaryotic cells (Holmgren, 1985). In higher plants, two thioredoxin systems are known. The first system consists of two chloroplastic thioredoxins (m and f). They are reduced by an Fd-dependent thioredoxin reductase and regulate photosynthesis by activating NADP-malate dehydrogenase (type m) and enzymes of the photosynthetic carbon cycle including Fru-1,6-biphosphatase (type f) (Johnson et al., 1987; Muller, 1991). The second system is reduced by an NADPH-dependent thioredoxin reductase. This enzymic activity has been characterized in nongreen heterotrophic tissues, and, consequently, the proteins have been named thioredoxin h (Johnson et al., 1987; Florencio et al., 1988). One spinach thioredoxin h has been purified and partially sequenced (Marcus et al., 1991). We recently isolated one tobacco cDNA clone (Marty and Meyer, 1991) and one genomic clone (Brugidou et al., 1993) encoding two thioredoxin h proteins. In this paper, we report the nucleotide and deduced amino acid sequences of an Arabidopsis thaliana thioredoxin h. A cDNA library from immature siliques of A. thaliana cv Columbia was constructed by J. Giraudat (Institut des Sciences Végétales at Gif-Sur-Yvette) in X Zap 11. The library was screened with a radiolabeled 509-bp cDNA clone encoding the tobacco thioredoxin h l . Of 150,000 plaques, 12 positive plaques were obtained from the first round of screening. The positive plaques were purified by further screening at lower density. After in vivo plasmidization, the clones were sequenced. A11 are identical, containing the same 497-bp insert (Fig. 1).The identity values (Table I) indicate that thioredoxin h proteins are related and show more homology with the vertebrate cytoplasmic thioredoxins than with the chloroplastic ones.
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Figure 1. Nucleotide and deduced amino acid sequences of the cDNA clone encoding thioredoxin h of A. thaliana. Noncoding regions are shown in lowercase. The disulfide active site i5 under-
lined. Nucleotides are numbered on the right, and amino acids are numbered on the left. ACKNOWLEDGMENTS
We thank Dr. J. Giraudat, Gif-Sur-Yvette, France, for the Arabi-
dopsis cDNA library, F. Grellet for help with DNA sequence analysis, and Dr. Richard Cooke for critica1 reading of the paper. Received September 29, 1992; accepted December 2, 1992. Copyright Clearance Center: 0032-0889/93/l02/0327/02. The GenBank accession number for the sequence reported in this article is 214084. LITERATURE ClTED
Brugidou C, Marty I, Chartier Y, Meyer Y (1993) The Nicotiana tabacutn genome encodes two cytoplasmic thioredoxin genes which are differently expressed. Mo1 Gen Genet (in press) Florencio FJ, Yee BC, Johnson TC, Buchanan BB (1988) An NADE' thioredoxin system in leaves: purification and characterization of NADE' thioredoxin reductase and thioredoxin h from spinach. Arch Biochem Biophys 2 6 6 496-507 Holmgren A (1985) Thioredoxin. Annu Rev Biochem 5 4 237-271 Johnson TC, Cao RO, Kung JE, Buchanan BB (1987) Thioredoxin
' R.R.-M. was supported by Consejo Nacional de Ciencia y Tecnologia, México. M.P. was supported by Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior, Brazil. * Corresponding author; fax 33-68-66-84-99. 327
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and NADP-thioredoxin reductase from cultured carrot cells. Planta 171: 321-331
Marcus F, Chamberlain SH, Chu C, Masiarz FR, Shin S, Yee BC, Buchanan BB (1991) Plant thioredoxin-h-an animal-like thioredoxin occurring in multiple cell compartment sources. Arch Biochem Biophys 287: 195-198 Marty I, Meyer Y (1991) Nucleotide sequence of a cDNA encoding a tobacco thioredoxin. Plant Mo1 Biol17: 143-147 Muller GD (1991) Thioredoxin deficiency in yeast prolongs S phase and shortens the G1 interval of the cell cycle. J Biol Chem 2 6 6 9144-9202 Pearson WR (1990) Rapid and sensitive sequence comparison with FASTAP and FASTA. Methods Enzymoll83: 63-93
Plant Physiol. Vol. 102, 1993
Table 1. Characteristics o f a cDNA encoding an A. thaliana thioredoxin h
Organism : Arabidopsis thaliana ecotype Colomhia. Sources: cDNA library in X Zap II constructed from poly(A)+ RNA of immature siliques. Isolation: The lihrary was screened with the radiolabeled 509-bp tobacco cDNA (GenBank X58527). Techniques of Sequencing and Sequence Identification: Double-stranded miniprep plasmid DNA sequencing with Sequenase; synthetic oligonucleotides to the known sequence used as primers; sequencing of both strands. Characteristics of cDNA: The 3’ untranslated region is 123 bp long, followed hy 15 bp of poly(A) tail. No obvious polyadenylation signal is present. Structural Features of the Deduced Amino Acid Sequence: Open reading frame of 115 amino acids, predicted molecular mass of the protein 12.6 kD. At position 43 to 48, it contains a sequence that perfectly matches the characteristic thioredoxin motif. Comparisons with other protein sequences using FASTA (Pearson, 1990) indicated homology with thioredoxin h clones (75% identity with tobacco H2 EMBL 211803, 65% identity with tobacco h l [GenPept S165901, 51.2% with spinach h thioredoxin [GenPept S151371, and 46.3% identity with Chlamydomonas thioredoxin h [SwissProt P80028]), with cytoplasmic eukaryote thioredoxins (45.7% identity with human [SwissProt P10599],41.9% with yeast [SwissProt P22803]), with prokaryote thioredoxin (37.7% with Escherichia coli [SwissProt POO274]), and with chloroplastic thioredoxins (f, 37.3% [SwissProt PO98561; m, 33.3% [SwissProt P075911). Method of Identification: Deduced amino acid sequence comparison with GenBank and EMBL data bases. Function: Reduction of disulfide bridges in polypeptides, involved in growth process.
