Abstract: Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is known to be an instrumental anionic phospholipid in governing pollen germination and pollen tube growth. However, the precise functions and regulatory mechanisms of PI(4,5)P₂ in pollen polarity establishment and germination remain poorly understood. Our previous studies demonstrated the pivotal involvement of Arabidopsis formin homology 5 (AtFH5)-dependent vesicle trafficking in polarity establishment of pollen. Here, we observed that PI(4,5)P₂ accumulated and oscillated at the prospective germination site, a process closely associated with the rotational movement of AtFH5-labeled vesicles. Disruption of the mobility of AtFH5-labeled vesicles, either through AtFH5 mutation or pharmacological treatment, significantly perturbed the accumulation of PI(4,5)P₂ at the plasma membrane. Subcellular localization and genetic analysis revealed that two phosphatidylinositol 4-phosphate 5-kinases, AtPIP5K1 and AtPIP5K4, are essential for PI(4,5)P₂ oscillation at the germination site prior to pollen germination. Furthermore, we found that the dynamics of AtPIP5K4 depended on the mobility of AtFH5-labeled vesicles and reduced PI(4,5)P₂ in turn disturbed the attachment of AtFH5-labeled secretory vesicles to the plasma membrane. In conclusion, these findings collectively highlight the reciprocal regulation of AtFH5-labeled secretory vesicles and PI(4,5)P₂ oscillations at the plasma membrane, providing critical insights into the molecular mechanism underlying polarity establishment during pollen germination.

Key words: polarity establishment, pollen germination, PI(4,5)P₂, secretory vesicles