PlantMolecular Biology 31: 553-564, 1996. @ 1996 KluwerAcademic Publishers. Printed in Belgium. 553 Primary structure and expression of plant homologues of animal and fungal thioredoxin-dependent peroxide reductases and bacterial alkyl hydroperoxide reductases Margarete Baier and Karl-Josef Dietz* Julius-von-Sachs-lnstitut fiir Biowissenschaften, Lehrstuhl fiir Botanik I, Mittlerer Dallenbergweg 64, 97082 Wiirzburg, Germany (*author for correspondence) Received 5 March 1996;accepted in revisedform 8 March 1996 Key words: cDNA-cloning, expression, Hordeum vulgare, leaf, peroxiredoxin, shoot development, Spinacia oleracea Abstract Higher plants express genes encoding peroxiredoxins of the two-cysteine type. This is concluded from the isolation of cDNAs from spinach (Spinacia oleracea) and barley (Hordeum vulgare cv. Gerbel) which are homologous to animal, fungal, and bacterial two-cysteine peroxiredoxins. Northern blot analysis indicated the presence of at least one corresponding gene in all angiosperms analyzed suggesting that basl is a member of an ubiquitous gene family encoding a protein of fundamental importance in oxidative stress defense also in plants. In barley, expression increased upon application of methyl viologen but was not affected by ozone, mRNA levels increased during deetiolation in the light. Maximal abundance of basl transcripts was observed in young developing shoot segments where cell division and elongation take place. Expression was insignificant in roots. The amount of basl protein was high in the leaf blade, particularly in etiolated plants, and did not respond to oxidative stress, bas I protein was not detected in roots. From our data, we suggest that basl is an antioxidant enzyme particularly important in the developing shoot and photosynthesizing leaf. Abbreviations: AhpC, C22 subunit of the alkyl hydroperoxide reductase; BSO, buthionine sulfoximine; DTT, dithiothreitol; EST, expressed sequence tag; TPx, thioredoxin-dependent peroxide reductase; SOD, CuZn superox- ide dismutase; TR, thioredoxin reductase; Trx, thioredoxin; TSA, thiol-specific antioxidant Introduction In recent years a new type of antioxidant enzyme has been found in animals and fungi [7, 8, 20, 23] designat- ed 'thioredoxin-dependent peroxide reductase' (TPx) [ 10]. Together with other animal proteins, such as pag [30] and MER5 [37], and bacterial alkyl hydroper- oxide reductases [33] it constitutes the two-cysteine sub-family of peroxiredoxins [8]. Jacobson et al. [18] and Chae et al. [10] showed that this enzyme reduces hydrogenperoxide and alkyl hydroperoxide, for instance t-butyl hydroperoxide The nucleotide sequence datareported will appearin the EMBL and NucleotideSequenceDatabases under the accession numbers X94219 (spinach)and Z34917(barley). and cumene hydroperoxide. In vitro, addition of thioredoxin-dependent peroxide reductase prevents damage of nucleic acids [22] and inactivation of enzymes [20] otherwise observed in the presence of Fe(III), 02 and thiols. Two cysteine residues are con- served in all homologous proteins belonging to the TPx/AhpC sub-family of peroxiredoxins [8]. Cova- lent modification with N-ethylmaleimide (MEM) [22] and site-specific mutagenesis [9] of C47 in yeast-TPx inactivate TPx suggesting that C47 is involved in the catalytic mechanism and functions as strong nucle- ophile [22]. According to the reaction mechanism pro- posed by Chae and coworkers [10] peroxides oxidize a sulfhydryl group in the reactive site of the enzyme. A disulfide bond is formed and links the two subunits