Genetic analysis of sperm cell formation in Arabidopsis thaliana L. Heynh

The genetics and cell biology of a novel class of mutants that affect microgametogenesis in Arabidopsis was investigated. These mutants termed duo pollen (duo) specifically block generative cell division preventing the formation of sperm cells. Genetic analyses revealed that duo1-duo6 gametophytically and are male-specific. The duo mutants mapped to different chromosomal locations. Phenotypic characterisation revealed that duo1-duo3 share similar but distant nuclear morphologies. Analysis of pollen grains exhibiting mitotic figures showed that the undivided generative cell in duo1 entered pollen mitosis II but failed to progress beyond prometaphase. In contrast duo2 and duo3 failed at G2-M transition of pollen mitosis II. The progression of S phase was monitored by measuring nuclear DNA contents throughout gametogenesis in the wild type and in duo1-duo3. In the wild type, sperm nuclei enter S phase soon after inception and continue DNA synthesis until anthesis. Moreover, the male gametic cells in wild type appear to follow a simple S to M cycle. In duo1 the generative nucleus completes DNA synthesis and maintains a 2C DNA value at anthesis. In contrast, mutant generative nuclei in duo2 and duo3 bypass pollen mitosis II and continue DNA synthesis during pollen maturation similar to sperm nuclei in the wild type. Map-based cloning defined the DUO3 locus to a 10 kb region. One of the genes, termed DUO3 successfully complemented the duo3 mutation. Nucleotide sequencing of DUO3 in heterozygous duo3 mutants revealed no sequence differences from the wild type, suggesting that DUO3 is an epiallele. DUO3 encodes a novel protein of 137 kDA containing a Myb-like domain known as the SANT domain. This work demonstrated gametophytic control that operates at G2-M and M phase of generative cell cycle. The identification of DUO3 gene provides a unique opportunity to understand the molecular basis of the generative cell division.