From a mid-maturation seed cDNA library we have isolated cDNA clones encoding two Triticum aestivum puroindolines. Puroindoline-a and puroindoline-b, which are 55% similar, are basic, cystine-rich and tryptophan-rich proteins. Puroindolines are synthesized as preproproteins which include N- and C-terminal propeptides which could be involved in their vacuolar localization. The mature proteins have a molecular mass of 13 kDa and a calculated isoelectric point greater than 10. A notable feature of the primary structure of puroindolines is the presence of a tryptophan-rich domain which also contains basic residues. A similar tryptophan-rich domain was found within an oat seed protein and a mammalian antimicrobial peptide. The ten cysteine residues of puroindolines are organized in a cysteine skeleton which shows similarity to the cysteine skeleton of other wheat seed cystine-rich proteins. Northern blot analysis showed that puroindoline genes are specifically expressed in T. aestivum developing seeds. No puroindoline transcripts as well as no related genes were detected in Triticum durum. The identity of puroindolines to wheat starch-granule associated proteins is discussed as well as the potential role of puroindolines in the plant defence mechanism.

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Two cDNA clones, pTaM13.38 and pTd14.13.2, encoding a Triticum aestivum and a Triticum durum thioredoxin h, respectively, were isolated from mid-maturation seed cDNA libraries. The T. aestivum thioredoxin h has a molecular mass of 13.5 kDa and that from T. durum has a molecular mass of 13.8 kDa. These two wheat thioredoxin h are 98.5% similar and contain the canonical WCGPC active site and the important structural and functional amino acids that are conserved in thioredoxin sequences. The recombinant T. aestivum thioredoxin h (TrxTa) overproduced in BL21(DE3)pLysS was purified to homogeneity by a three-step procedure including heat treatment, anion-exchange chromatography and gel filtration. TrxTa showed a lower stability to high temperature than Escherichia coli thioredoxin or plant thioredoxin m. The molecular mass of TrxTa, determined by mass spectrometry, is 13 391 Da and corresponds to a protein lacking the first methionine residue, as confirmed by its N-terminal end sequence AASAAT. Using the 5,5′-dithiobis(2-nitrobenzoic acid)-reduction assay and monobromobimane revelation we showed that TrxTa is specifically reduced by wheat NADP:thioredoxin reductase (NTR), and not by E. coli NTR. TrxTa is able to reduce identified target proteins i.e. wheat seed A-amylase inhibitors (chloroform/methanol-soluble proteins). The presence of a putative transmembrane domain at the N-terminal end of the two wheat thioredoxins raises the question of whether these proteins are membrane anchored.

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