Intron Position as an Evolutionary Marker of Thioredoxins and Thioredoxin Domains Mariam Sahrawy, 1 Vale ´rie Hecht, 2 Javier Lopez-Jaramillo, 1 Ana Chueca, 1 Yvette Chartier, 2 Yves Meyer 2 1 Department of Plant Biochemistry, Estacion Experimental del Zaidin, Consejo Superior de Investigaciones, Cientificas, Professor Alberada 1, 18008-Granada, Spain 2 Laboratoire de Physiologie et Biologie Mole ´culaire des Plantes, Universite ´/Centre National de la Recherche Scientifique URA, 565 Avenue de Villeneuve, 66860 Perpignan, Cedex, France Received: 14 September 1995 / Accepted: 4 November 1995 Abstract. In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequenced Arabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently pub- lished sequence of Chlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA ge- nomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this se- quence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In con- trast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomer- ases. This strongly supports the hypothesis of a eukary- otic origin for chloroplastic thioredoxin f. Key words: Thioredoxins — Introns — Phylogeny Introduction Thioredoxins are proteins (approximately 100–120 amino acids) implicated in various oxidoreductive reac- tions. They contain an active site with the conserved dicysteine sequence of WCxxC able to form a disulfide bridge in the oxidized state (Holmgren 1985). Thiore- doxins have been characterized in all pro- and eukary- otes. It is generally assumed that thioredoxins are disul- fide reductases able to break disulfide bridges of target proteins, but they may function to a lesser extent in the opposite way, promoting disulfide bridge formation, at least in vitro. Prokaryotes and vertebrates typically pos- sess only one thioredoxin gene per genome, while fungi contain multiple but closely related thioredoxins. The situation is far more complex in higher plants where three different thioredoxin types have been character- ized. Thioredoxins h have no transit peptide (Marty and Meyer 1991) and are consequently assumed to be cyto- plasmic; nevertheless a rice thioredoxin h has been de- tected as a major phloem sap protein (Ishiwatari et al. 1995). We have recently shown that Arabidopsis thaliana encodes at least five different thioredoxins of Correspondence to: Y. Meyer J Mol Evol (1996) 42:422–431 © Springer-Verlag New York Inc. 1996