Cellular and genetic control of male germline morphogenesis in Arabidopsis thaliana

The development of the generative cell (GC) in angiosperm pollen involves a unique morphogenesis with two keys features, first, the formation of a long cytoplasmic extension which physically connects the vegetative nucleus in a structural assemblage known as the male germ unit (MGU), and second, the body of the GC in Arabidopsis undergoes progressive morphogenesis from lens-shaped to round, fully elongating before dividing to form two sperm cells. The genetic and molecular mechanisms involved in germline morphogenesis are unknown and it was hypothesised that the male germline-specific transcription factor, DUO1 might regulate one or both structural features. Generative cell morphogenesis was examined by differential interference contrast and fluorescence microscopy of introduced nuclear and cytoplasmic markers in gametophytic mutants affecting GC division. In four allelic duo1 mutants the body of the GC fails to undergo elongation, but cytoplasmic extensions of the GC body are clearly established. It was hypothesised that DUO1 might regulate morphogenesis through any of its direct target genes and/or through components of the DUO1-DAZ1 network. Developmental analysis was performed in plants harbouring T-DNA insertions in seven known DUO1 target genes and it was found that only pollen carrying mutations in DAZ1 and DAZ2 show abnormal GC morphogenesis. The cytoplasmic extension of the generative cells and sperm cell pair is thought to play an important role in the establishment of the MGU in angiosperms, while the underlying cellular and molecular mechanisms are unknown. It was hypothesised that the physical association between the male germ cells and vegetative nucleus might be disturbed or lost in germ unit malformed (gum) mutants. In vivo microscopic analysis demonstrated that physical linkage of the cytoplasmic extension with the VN appear intact in gum mutants irrespective of the apparent increased distance between the VN and germ cell nuclei.