Two promoters conferring active gene expression in vegetative nuclei of tobacco immature pollen undergoing embryogenic dedifferentiation

In order to visualize the specific state of tobacco pollen undergoing dedifferentiation from immature pollen to embryogenic cells, we established tobacco marker lines transgenic for a vital reporter gene regulated under the transcriptional control of an 840 bp fragment, named A22pro. This fragment was obtained from the 5'-flanking region of a gene corresponding to a cDNA named A22 that was previously isolated through differential screening from a cDNA library prepared from tobacco pollen undergoing dedifferentiation. The reporter gene, named H3sGFP, consisting of synthetic green fluorescent protein gene (sGFP) and tobacco H3 histone gene for nuclear localization, was designed to distinguish the gene expression in the generative cell from that in the vegetative cell in a pollen grain. The marker line produced pollen showing a green fluorescent signal in the generative nuclei (GN) but the expression level of the transgene was low. Pollen after culture for dedifferentiation showed an intense signal transiently in the vegetative nuclei (VN), at a specific developmental stage of pollen, with a rapid increase of expression level of the transgene. Serial observations revealed that all androgenic embryos originated from the pollen grains that had shown the signal in their VN. Thus, A22pro is originally functional in gametogenesis but is activated in VN of pollen undergoing embryogenic dedifferentiation. Additionally, we observed a gene expression pattern identical to that described above, using another 5'-flanking region of a gene for a cDNA, named B27pro, homologous to A22 as a promoter of the reporter gene.