Characterization of two thioredoxins h with predominant localization in the nucleus of aleurone and scutellum cells of germinating wheat seeds

Two full-length cDNA clones, designated TrxhA and TrxhB, encoding different but very similar thioredoxin h polypeptides were isolated from wheat (Trit icum aestivum cv. Chinese Spring) aleurone cells. The deduced proteins show a high similarity to each other and to thioredoxin h from other sources, i n particular from T. aestivum and T. durum. One of them, TRXhA, was express ed in E. coli as a His-tagged polypeptide and used to raise polyclonal anti bodies by immunization of rabbits. These antibodies identified a single ban d (ca. 13.5 kDa) in western blot analysis of protein extracts from all whea t organs analyzed. TRXhA and TRXhB when expressed in E. coli as intact poly petides showed indistinguishable electrophoretic mobility, which correspond ed to the 13.5 kDa polypeptide detected in wheat protein extracts. The amou nt of thioredoxin h transcripts increased in scutellum and aleurone cells d uring germination but GA(3) did not exert any stimulatory effect on thiored oxin h expression. Although northern blot analysis detected a single band, competitive RT-PCR showed that this band is due to the accumulation of both TrxhA and TrxhB mRNAs. These results suggest that the single band detected in western blots is due to the presence of at least two thioredoxin h poly peptides. Immunolocalization experiments confirmed the high content of thio redoxins h in scutellum and aleurone cells, and showed a low content in the starchy endosperm of germinating grains. Interestingly, though these prote ins are evenly distributed in the cytosol, the highest levels of thioredoxi ns h were detected in the nucleus, both in aleurone and scutellum cells.