Isolation and characterization of male-germ-cell transcripts in Nicotiana tabacum

Abstract.

Until recently, little knowledge existed about the molecular mechanisms regulating male gamete development. This was mainly due to the low transcriptional activity and the cellular inaccessibility of the generative and sperm cells that are enclosed by the vegetative cell in pollen. In order to study sperm cell development and possible preferential fusion during double fertilization, we have constructed a cDNA library of mRNA isolated from pure tobacco sperm cells. An initial screen of 396 clones from this library has yielded 2 cDNAs representing sperm-cell-expressed transcripts, designated NtS1 and NtS2. A preliminary characterization of these two clones showed that they accumulate in both the generative and sperm cells (i.e. the male gamete) indicating that gene expression programs between these two cell types overlap. In addition, we found that NtS1 codes for a polygalacturonase suggesting a role for this enzyme in wall degradation during differentiation of the male germ cells in tobacco. Together, these results show that with the construction of this sperm-cell cDNA library we now have a powerful tool to investigate male gamete development and function.