Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth

Bloch D.*, Pleskot R.*, Pejchar P.*, Potocký M., Trpkošová P., Cwiklik L., Vukašinović N., Sternberg H., Yalovsky S.‡, Žárský V.‡ *These authors contributed equally to this work ‡Corresponding authors
PLANT PHYSIOLOGY 172 (2): 980-1002, 2016

Keywords: Arabidopsis, exocyst, phosphoinositides, pollen tube, tip growth, tobacco
Abstract: Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle tethering complex functions in polarized exocytosis. Here we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The GFP-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in development of pollen with multiple tips, indicating that SEC3 is required for determining the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a expressing pollen with the endocytic marker FM4-64 revealed the presence of sub-domains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco pollen tubes, SEC3a displayed N-terminal PH-like domain (SEC3a-N) dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and function of SEC3a in Arabidopsis. Taken together, our findings indicate that SEC3a is critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery pending on pollen tube growth modes.
DOI: ​10.​1104/​pp.​16.​00690 IEB authors: Přemysl Pejchar, Roman Pleskot, Martin Potocký, Nemanja Vukasinovic, Viktor Žárský