The correct development of male gametophytes (pollen grains) in flowering plants is essential for proliferate in gamete production. Here we have taken a map-based cloning approach using Arabidopsis male gametophytic mutant, named gemini pollen3 (gem3) to identify and characterize key gene that is expressed gametophytically for the completion of microgametogenesis focusing on genes which control cell division and cell fate determination. Previously reported gem1 and gem2 mutants with similar characteristics to gem3 that are disturbed at asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. However, gem3 was mapped to a different genetic locus, and pollen developmental analysis revealed that gem3 exert an effect at meiosis and mitosis causing complete sterility. We also discovered that gem3 homozygous lines produce aberrant pollen grains, arising from incomplete cytokinesis during male meiosis with sporophytic phenotypes of twisted-shape leaves, large flowers. This mutation shows reduced genetic transmission of gem3 allele through male gametophyte. In previous results, the gem3 locus was confirmed by mapping to the region located on chromosome 5. To further confirm strong candidate gene, we performed sequencing and genetic complementation analysis. Currently, we are performing functional studies of the gem3 gene for the better understanding of molecular mechanisms that control asymmetric division at meiosis and mitosis during pollen development.

Arabidopsis Fused kinase TWO-IN-ONE (TIO) controls phragmoplast expansion and interacts with the Kinesin-12 subfamily proteins that anchor the plus ends of interdigitating microtubules (MTs) in the phragmoplast midzone. Previous analyses of loss-of-function mutants and RNA interference lines revealed that TIO positively controls both somatic and gametophytic cell cytokinesis, however, knowledge of the full spectrum of TIO functions during plant development remains incomplete. In order to further characterize TIO functions, we expressed TIO and a range of TIO variants under control of its own promoter in wild type Arabidopsis plants. We discovered that TIO-overexpressing transgenic lines produce enlarged pollen grains, arising from incomplete cytokinesis during male meiosis, and showed sporophytic abnormalities indicating polyploidy. These phenotypes arose independently in TIO variants that abolished either gametophytic function or the ability of TIO to interact with Kinesin-12 subfamily proteins. Interaction assays in yeast showed TIO to bind to AtNACK2/TETRASPORE and plants doubly homozygous for kinesin-12a and kinesin-12b knockout mutations to produce enlarged pollen grains. Our results show that TIO dominantly inhibits male meiotic cytokinesis in a dosage dependent manner that may involve direct binding to acomponent of the canonical NACK-PQR cytokinesis signaling pathway.