| Abstract: | Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.Plant development and growth require coordinated tissue and cell polarization. Two of the most essential cellular processes involved in polarization are cell expansion and cytokinesis, which determines cell morphology and functions (Jaillais and Gaude, 2008; Dettmer and Friml, 2011; Li et al., 2012). Pollen tube and root hair growth require highly polarized membrane trafficking (Libault et al., 2010; Kroeger and Geitmann, 2012). Cytokinesis, by which new cells are formed, separates daughter cells by forming a new structure within the cytoplasm termed the cell plate (CP). Made up of a cell wall (CW), surrounded by new plasma membrane (PM), the cell plate is generally considered to be an example of internal cell polarity in a nonpolarized plant cell (Bednarek and Falbel, 2002; Baluska et al., 2006).The conventional view of pollen tube tip growth and cell plate formation is supported by polar exocytic secretion of numerous vesicles (diameter of 60–100 nm) to the pollen tube tip and phragmoplast areas during cytokinesis. These polar exocytic vesicles, which are generally believed to originate from the Golgi apparatus, are delivered to the site of secretion via the cytoskeleton and fuse with the target membrane with the aid of fusion factors (Jurgens, 2005; Backues et al., 2007). However, whether these polar exocytic vesicles undergoing post-Golgi trafficking are part of the conventional Golgi-trans-Golgi network (TGN)-PM/CP exocytosis or are derived from some other unidentified exocytic secretion pathway remain unclear.Polar exocytosis is regulated and controlled by a conserved Rho GTPase signaling network in fungi, animals, and plants (Burkel et al., 2012; Ridley, 2013). Rho of plant (ROP), the sole subfamily of Rho GTPases in plant, participate in signaling pathways that regulate cytoskeleton organization and endomembrane trafficking, consequently determining cell polarization, polar growth and cell morphogenesis (Gu et al., 2005; Lee et al., 2008). In growing pollen tubes, ROP1 participates in regulating polar exocytosis in the tip region via two downstream pathways to regulate apical F-actin dynamics: RIC4-mediated F-actin polymerization and RIC3-mediated apical actin depolymerization. A constitutively active mutant of ROP1 (CA-rop1) prevents fusion of these vesicles with the PM and enhances the accumulation of exocytic vesicles in the apical cortex of pollen tubes (Lee et al., 2008). Although ROP GTPases have been extensively researched, their roles in polar membrane expansion in pollen tubes and epidermal pavement cells remains unclear (Xu et al., 2010; Yang and Lavagi, 2012), and there have been insufficient studies on the functions of ROPs in controlling cell plate formation during cytokinesis. Cell division requires precise regulation and spatial organization of the cytoskeleton for delivery of secretion vesicles to the expanding cell plate (Molendijk et al., 2001).In addition, newly made cell walls during cell expansion and cell plate formation require sufficient plasticity in order to integrate new membrane materials to support the polarized membrane extension. They also should be strong enough to withstand the internal turgor pressure and thereby maintain the shape of the cell (Zonia and Munnik, 2011; Hepler et al., 2013). Recent studies have demonstrated that pectins are important for both cytokinesis and cell expansion (Moore and Staehelin, 1988; Bosch et al., 2005; Chebli et al., 2012; Altartouri and Geitmann, 2015; Bidhendi and Geitmann, 2016). Pectins are one of the major cell wall components of the middle lamella and primary cell wall. They are polymerized and methylesterified in the Golgi and subsequently released into the apoplastic space as “soft” methylesterified polymers. The homogalacturonan components of pectin are later de-methylesterified by pectin methylesterases (PMEs). The demethylesterified pectins can be cross-linked, interact with Ca2+, and finally form the “hard” pectin matrix of the cell wall. Therefore, the enzymatic activity of PMEs determines the rigidity of the cell wall (Micheli, 2001; Peaucelle et al., 2011).In Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) pollen tubes, PMEs are found predominantly polar localized in the tip region and determine the rigidity of the apical cell wall (Bosch et al., 2005; Jiang et al., 2005; Fayant et al., 2010; Chebli et al., 2012; Wang et al., 2013). PME isoform knockout mutants in Arabidopsis (AtPPME1 or vanguard1) produce unstable pollen tubes which burst when germinated in vitro and have reduced fertilization abilities (Jiang et al., 2005; Rockel et al., 2008). Recent studies have shown that in growing tobacco pollen tubes, polar targeting of NtPPME1 to the pollen tube apex depends on an apical F-actin mesh network (Wang et al., 2013). Although the functions of PME in cell wall constriction are well documented, the intracellular secretion and regulation mechanism of the exocytic process of PME still remain largely unexplored. In addition, pectins are also found to be abundant in the forming cell plate, raising the possibility that PMEs may also function during cell plate formation (Moore and Staehelin, 1988; Dhonukshe et al., 2006).In our study, we have used NtPPME1 as a marker to identify a polar exocytic process which is distinct from the conventional Golgi-TGN-PM exocytosis pathway in both pollen tube tip growth and cell plate formation. We have identified a Golgi-derived secretory vesicle (GDSV) for the polar secretion and targeting of NtPPME1 to the cell wall that bypasses the TGN during cell polarization. Further investigations using ROP1 mutants have shown that this polar exocytosis is ROP1 dependent. |
