The Roles Vacuolar Sorting Receptor (VSR) and Secretary Carrier Membrane Protein (SCAMP) in Pollen Germination

The plant endomembrane system contains several membrane-bound organelles including the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN) or early endosome (EE), prevacuolar compartment (PVC) or late endosome (LE). In tip-confined rapid growing pollen tube, delivery of cell membrane and wall constructional materials to fast expanding tip region requires spatial and temporal coordination of secretory vesicle trafficking and endocytosis. Thus, polarized and rapid growing pollen tube is regarded as an excellent single cell model system to study molecular mechanisms of protein trafficking, vesicular secretion and endocytosis in plant cells. In this thesis research, I have made the following accomplishments in studying how pollen regulate and co-ordinate exocytosis and endocytosis processes to achieve rapid and polarized growth. Firstly, I have tested and optimized a pollen grain transient expression procedure for pollen grains of three commonly used plant species including lily, tobacco and Arabidopsis for studying protein subcellular localization and organelle dynamics. Secondly, using newly cloned lily vacuolar sorting receptor (VSR) and secretory carrier membrane protein (SCAMP) cDNA (termed LIVSR and LISCAMP respectively) as well as specific antibodies against VSR and SCAMP1 as tools, I have demonstrated that in growing lily pollen tubes (i) transiently expressed GFP-VSR/GFP-LIVSR located throughout the pollen tubes except the apical clear zone region whereas GFP-LISCAMP was mainly concentrated in the tip region; (ii) VSRs are localized to multivesicular body (MVB) and vacuole, while SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LIVSR or LISCAMP siRNAs significantly reduced the growth rate of the lily pollen tubes. Taken together, these results demonstrate that both VSRs and SCAMPs are essential for pollen tube growth, likely working together coordinately in the secretory and endocytic pathways respectively.