Cloning and characterization of three thioredoxin h isoforms from wheat showing differential expression in seeds

Plants contain several genes encoding thioredoxin h. In cereals, type-h thioredoxins are abundant in developing and germinating grains, but the mechanism regulating the expression of these genes and their specific function is poorly known. The cloning of three full-length cDNAs encoding thioredoxin h, stated Trxh1, Trxh2 and Trxh3, from wheat (Triticum aestivum cv. Soissons) seeds is described here. TRXh2 and TRXh3 deduced proteins show high identity between them and with other thioredoxins h previously described from wheat, and contain exclusively the two Cys residues forming part of the active site. By contrast, TRXh1 shows a lower level of identity and contains an additional Cys residue. The three wheat thioredoxins were expressed in E. coli and their activity was demonstrated using both the DTT-dependent insulin assay and a coupled assay with recombinant NTR from wheat. Site-directed mutagenesis showed that the additional Cys residue of TRXh1 has a low effect on its activity but is essential for dimerization. Specific expression of the three thioredoxin genes was analysed by real-time RT-PCR in developing and germinating seeds and seedlings under stressed and unstressed conditions. An increase of Trxh1, Trxh2, and Trxh3 transcripts was detected at the beginning of the desiccation phase during seed development. Early after imbibition, Trxh1, but not Trxh2 or Trxh3, transcripts showed a transient increase. Treatment of wheat seedlings with salt or hydrogen peroxide caused a differential pattern of expression of the three Trxh genes between and within tissues, hence suggesting specific functions for these thioredoxins during germination and early seedling growth.