Cloning of cDNA encoding NtEPc, a marker protein for the embryogenic dedifferentiation of immature tobacco pollen grains cultured in vitro

We partially purified three Nicotiana tabacum L. embryogenic pollen-abundan t phosphoproteins (NtEPa to c) which appeared in the cells undergoing a ded ifferentiation process from immature pollen grains to embryogenic cells, ca used by glutamine-deficiency in vitro, All the NtEPs had a highly conserved N-terminal amino acid sequence, Using degenerate oligonucleotide probes de signed from the amino acid sequences, the cDNA for NtEPc was isolated from a cDNA library of pollen cultured in glutamine-free medium. The cDNA sequen ce showed moderate homology with several type-1 copper-binding glycoprotein s and with a kind of early nodulin though its function could not be predict ed, Expression analysis revealed that the level of mRNA for NtEPc was high during the dedifferentiation and also in the very early period of pollen em bryogenesis but it was low in the developmental process of microspores/poll en in anthers, in the in vitro maturation process and both in the stational and logarithmic growth phases of tobacco BY-2 cells. Furthermore, an acidi c medium pH, which promoted the induction of dedifferentiation increased th e level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, whi ch inhibited it, decreased the level. These results suggest that the expres sion of NtEPc gene is correlated with the dedifferentiation but not with po llen development or cell division.