Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana.

Abstract

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...

A Novel Type of Thioredoxin Dedicated to Symbiosis in Legumes

“…With Trx s1r, the reductase exhibits an apparent K m of 13.8 6 2.1 mM with a k cat of 0.27 6 0.02 s 21 . The K m value obtained is similar to that calculated for Trx h2r (13.0 6 1.1 mM) and is in the range of those determined for NTR and Trxs h and m from Arabidopsis and Chlamydomonas reinhardtii (Rivera-Madrid et al, 1995;Stein et al, 1995). However, the k cat value determined for Trx s1r is 5-fold lower than that measured for Trx h2r (1.47 6 0.05 s 21 ), indicating that MtNTRA reduces Trx s1r less efficiently than Trx h2r.…”

Thioredoxins: structure and function in plant cells

“…This protein shows only c. 35 % homology to all thioredoxin types known so far (mammalian, m, h or/) which probably explains why its gene has not been detected yet in other animal cells where only two genes have so far been identified, one of which is non-productive (Tonissen & Wells, 1991;Kaghad et al, 1994). Interestingly, in plants at least one and possibly several thioredoxin h show extended N-terminus sequences (Rivera-Madrid et al, 1995), but it is not known whether this represents a signal peptide and whether these proteins are really imported into the mitochondria. Also it is not known whether mitochondria contain a thioredoxin reduction system (namely NADPH -I-thioredoxin reductase).…”

“…Thioredoxins h are far more variable with a surprisingly' high number of sequences in a single organism: five complete sequences in the case of A. thaliana (Rivera-Madrid et al, 1995), and three additional expressed sequence tags (EST) (not shown). As evidenced by the tree, the divergence within the A. thaliana thioredoxin h sequences is old, probably preceding the divergence between monocots and dicots.…”

“…Again, this situation is reminiscent of the regulatory effects of human thioredoxin on the transcription factors NF Kappa B and API and of the anti-oxidative stress properties of thioredoxin reported in bacteria and yeast. In tobacco and A. thaliana, several cDNAs have been isolated when screening for mRNAs strongly expressed in actively dividing tissues (Marty & Meyer, 1991 ;Brugidou et al, 1993;Rivera-Madrid et al, 1995). In addition, a tefl box (an ubiquitous cis-acting element involved in the activation of plant genes that are highly expressed in cycling cells) has been found in the promoter region oi A. thaliana thioredoxin h (Regad et al, 1995) and it is also present in the 5' region of the C. reinhardtii thioredoxin h gene (Stein et al, 1995 and unpublished).…”

A Novel Nuclear Member of the Thioredoxin Superfamily

“…An active-site motif of WCGPC is conserved among the classic TRXs of E. coli and many other species. However, other active-site sequences exist, including a WCPPC motif that has been identified in TRX and TRX-like sequences within diverse organisms such as Arabidopsis, Caenorhabditis elegans, and Mus musculus (Rivera-Madrid et al, 1995;Kurooka et al, 1997). The classic TRX has its active site protruding from a highly organized, globular structure composed of five strands of ␤-sheets enclosed by four ␣-helices (Holmgren, 1985).…”