The Tip-localized Phosphatidylserine Established By Arabidopsis ALA3 Is Crucial For Rab GTPAse-Mediated Vesicle Trafficking And Pollen Tube Growth

Pollen tube growth is an indispensable event during double fertilization in flowering plants.It is a typical apical polar growth mode,which relies on accurate"transport system"to ensure its rapid tip growth.Rab GTPases are known as a key class of conserved proteins responsible for vesicle trafficking in eukaryotes,participating in a variety of endomembrane trafficking processes:vesicle budding,transportation,tethering,and membrane fusion.Additional evidence of the relationship between Rab GTPases and pollen development and polarized pollen tube growth has been obtained from research on Arabidopsis Rab A and Rab D.In particular,RFP-Rab A4d-labeled vesicles are specifically distributed at the apical region of the pollen tube,but the mechanism involved in regulating its polarity distribution has not been reported.In eukaryotic cells,anionic phospholipids,including phosphatidylinositol phosphates?PIPs?,phosphatidylserine?PS?,and phosphatidic acid?PA?contribute to the surface charge,curvature and lipid packing of the membrane can effectively control the localization and activity of Rab GTPases.Among these phospholipids,phosphatidylinositol-4-phosphate?PI4P?,phosphatidylinositol-4,5-phosphate?PI?4,5?P2?and PA are three well-studied anionic lipids that localize to the plasma membrane?PM?of pollen tubes,but another lipid,PS,is mainly enriched in the inverted-cone zone of the tobacco pollen tube apex,suggesting that PS might be involved in Rab GTPases mediated vesicle trafficking in the apical area of pollen tubes.In addition,the asymmetric distribution of PS in plant cells depends on P4-ATPases?Aminophospho Lipid ATPases?ALAs??,which flip PS across the biomembrane from the extracellular/luminal leaflet to the cytosolic leaflet powered by ATP hydrolysis in eukaryotes.In recent years,progress in the research on flippases in plants has shown that P₄-ATPases have pleiotropic effects on physiological functions,including plant growth and development,reproduction,and adaptation to biotic and abiotic stress.However,whether ALAs are involved in the establishment of the asymmetric distribution of PS in pollen tubes remains unclear.Further research is also required to clarify the subcellular localization and biological function of PS in pollen tubes.In this study,we functionally characterized Arabidopsis ALA3 and ALA3 established PS in pollen tube tip growth using biochemical,cytobiology and genetic evidences,and the mechanism of phosphatidylserine?PS?regulated pollen tube tip growth by promoting vesicle polar transport was discussed.1.The role of ALA3 in pollen tube tip growth was revealed.Loss of ALA3function not only inhibited the pollen tube growth,but also increased the width of pollen tube and decreased the sensitivity to BFA in vitro.Exogenous ALA3 could completely complement the phenotype of pollen tube width and partially complement the phenotype of pollen tube length,while the exogenous ALA3 without phospholipid flipping activity(ALA3^D413N)could not complement the pollen tube phenotypes of ala3,suggesting that ALA3 regulates the polar growth of pollen tube through its conserved phospholipid flipping activity.2.ALA3 was strongly expressed in pollen and pollen tubes,and loss of ALA3function results in apical vesicles labeled by FM4-64,YFP-Rab A4b and RFP-Rab A4d decreased at the pollen tube apical zone and accumulated at the subapical zone,suggesting that ALA3 is required for vesicle polar transport.3.ALA3 was abundantly distributed in the apical region of pollen tube.The co-localization analysis of ALA3-GFP and various organelle labeled proteins fused with fluorescent tags showed that ALA3 was mainly located in the apical plasma membrane,TGN/EE,post-Golgi apparatus and recycling endosome of the pollen tube.4.Genetic evidence demonstrated that ALA3 and Rab A4d collectively regulated the polar growth of pollen tubes.The width of pollen tube of ala3 raba4d double mutant was not significantly increased compared with that of the single mutants,and the polarity distribution of vesicles in raba4d pollen tube was lost and the number of vesicles decreased significantly,which was similar to that of ala3.5.Loss of function of ALA3 alters the distribution pattern and the amount of PS in pollen tubes.The co-localization analysis of PS sensor EGFP-Lact-C2 and organelle labeled proteins fused with fluorescent tags in the pollen tube showed that the distribution pattern and subcellular localization pattern of PS was similar to that of ALA3-GFP;loss of function of ALA3 led to the polarized distribution of PS dramatically decreased in the apical region of pollen tube,chiefly accumulating in the subapical region;in vitro biochemical results showed that PS could bind to various forms of Rab GTPases.6.ALA3 controls vesicle formation and polar transport by regulating the formation of TGN/EE and the localization and distribution of secretory Rab GTPases,thus regulating the deposition of cell wall components and finally affecting the tip growth of pollen tube;ALA3 probably affects the binding of PS with various forms of Rab GTPases by regulating the distribution of PS in pollen tube and then affecting the distribution and activity of Rab GTPases.