Isolation of cDNAs coding for NtEPb1-b3, marker proteins for pollen dedifferentiation in a tobacco pollen culture system

Several phosphoproteins, Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPs), characteristically appear in the dedifferentiation process from immature pollen grains to embryogenic cells in a pollen culture system. Among NtEPs we focused our attention on three proteins (NtEPb1-b3) which showed the highest correlation with the dedifferentiation and possessed different pI values and similar molecular weights (ca. 22 kDa). Using probes designed from the N-terminal amino acid sequence common to the three, we isolated 14 clones of cDNA belonging to three similar sequences which probably correspond to NtEPb1-b3. The predicted amino acid sequences showed moderate homology to NtEPc, several type-1 copper-binding glycoproteins and a kind of early nodulin. The level of the transcripts for NtEPbs is highly associated with the pollen dedifferentiation but not with pollen maturation nor with cell division accompanied by meiosis or proliferation of BY-2 cells. Such an expression manner was distinguished from that of a gene coding for A-type cyclin (Ntcyc 25), indicating that NtEPb genes are not under cell cycle control. These results suggest that there exist genes related to an unknown event other than the reentrance of cell cycle in the dedifferentiation process of immature pollen that may be important for acquisition of embryogenic competence.