Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP+-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes. Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in theArabidopsis genome. This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation. This diversity allows the transduction of a redox signal into multiple pathways.Thioredoxins are small proteins (105-120 aa) that contain the strictly conserved sequence Trp-Cys-Gly-Pro-Cys (1). The reduced cysteine pair forms a very reactive center able to disrupt the disulfide bridge of target proteins. In vivo, thioredoxins are reduced by a thioredoxin reductase. Thioredoxins have been isolated from prokaryotes and eukaryotes, including vertebrates, invertebrates, and fungi (2, 3, 41). Higher plants present two independent thioredoxin systems: the chloroplastic system consists of two nuclear gene-encoded proteins, thioredoxins m and f, which are reduced by a ferredoxindependent thioredoxin reductase (4, 5), and regulates the dark/light-related cycles (6, 7). In addition, the presence of a thioredoxin h system that is reduced by an NADP-dependent thioredoxin reductase was demonstrated some years ago in heterotrophic plant tissues (8, 9), but the first sequences for plant thioredoxins h (10-13) and NADP-dependent thioredoxin reductase (14) were obtained only recently. In this paper, we show that the Arabidopsis thaliana genome encodes at least five very divergent thioredoxin h genes, § a situation similar to the one of Dictyostelium discoideum (15) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Toulouse) were subcultured every 2 weeks by 10-fold dilution in B5 medium (42) with sucrose (20 mg/ml) and 2,4-dichlorophenoxyacetic acid (1 ,ug/ml) and collected after 3 days of subculture (growth phase). Primary calli (seeds germinated on the callus medium) and established calli (the cell suspension grown on the callus medium) were grown for 1 month on the same medium solidified with 0.8% agar. In vitro plantlets were obtained from seeds germinated and cultivated in vitro for 1 month on the callus medium lacking 2,4-dichlorophenoxyacetic acid. Mesophyll pr...