Investigation of pollen cell fate determination in Arabidopsis thaliana

The aim of the work is to investigate how asymmetric cell division and cell lineage isolation are linked to pollen cell fate determination. This was analysed by monitoring the activation of vegetative and male germline-specific markers in mutants, which disturb microspore polarity and division asymmetry (gemini pollen 1), or which disturb cytokinesis and cell lineage isolation (two-in-one). In twin-celled equally divided pollen present in late bicellular and mature anthers, the activation of VCK promoter was expressed in both daughter cells, while the activation of DUO1 and HTR10 promoters were not expressed at any stages. The centromeric chromatin fate marker HTR12-GFP is present at discrete nuclear foci in generative cell and sperm cell nuclei in wild type pollen, while in gem1 equal daughter cells the marker was initially detected in foci, but later was dispersed. Further, transposable elements activation represented by GUS reporter activity was present in wild type, twin-celled and binucleate pollen. These results support the hypothesis that asymmetric division of the microspore is required for the specification of generative cell fate and differential centromere identity between vegetative and generative cell nuclei. The analysis of cell fate in tio mutants showed that isolation of the germ cell lineage is not required for the initial activation of generative cell fate, but is essential for the maintenance of differential centromere identity. In binucleate tio pollen, generative cell fate markers were expressed despite the failure to partition daughter nuclei into separate cells. Three different cell fate categories were observed among binucleate tio pollen, including pollen with vegetative cell fate only, generative cell fate only and pollen with mixed cell fate. Moreover, the centromeric fate marker was initially present at discrete nuclear foci in both nuclei of binucleate tio pollen and later was dispersed. In a study of MICROTUBULE ORGANIZATION1 (MOR1) fused to YFP, the protein was localised as a perinuclear signal in microspores and enriched in the male germline of wild type pollen, while MOR1-YFP was not expressed in mutant gem1 and tio pollen.