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Exocytosis is a fundamental process for cells of all eukaryotic organisms. It is required for the secretion of extracellular materials and for the enlargement of the plasma membrane. In plant cells, exocytosis is also crucial for cell wall formation and cell elongation, which are coupled processes. Vesicle fusion for anisotropic plant cell elongation occurs in a non-random pattern, which implies that the sites of exocytosis must be temporally and spatially controlled. This could occur through directed Golgi body transport by the actin cytoskeleton and the targeting of Golgi vesicles to specific sites of the plasma membrane and/or by tethering and docking of the vesicles to a specific membrane domain before exocytosis. In yeast, the exocyst is involved in vesicle tethering before exocytotic vesicle membrane fusion with the plasma membrane. As almost all plant cells expand in a polar fashion, by either axial or tip growth, a local tethering complex could be important for the determination of the orientation of cell expansion.
In budding yeast, bud growth requires polarized exocytosis. An octameric protein complex, termed the exocyst, has been identified that serves as a tethering factor for exocytotic vesicles in the bud tip (TerBush et al., 1996). Polarized localization of the exocyst is essential for polarized secretion during bud formation (Hsu et al., 2004; Munson & Novick, 2006; Zhang et al., 2008; Songer & Munson, 2009).
The exocyst consists of SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70 and EXO84 (Hsu et al., 1996; Kee et al., 1997; Eliás et al., 2003; Li et al., 2010; Zhang et al., 2010). Budding yeast SEC3 is considered to be a landmark protein for polarized exocytosis, as it is localized to the plasma membrane at which exocytosis will occur and is involved in the recruitment of the other exocyst subunits that reside in the cytoplasm or are associated with vesicles at this location (Finger et al., 1998; Wiederkehr et al., 2003; Hutagalung et al., 2009). It is thought that the polarized localization of SEC3 is mediated by interactions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the inner leaflet of the plasma membrane (He et al., 2007; Liu et al., 2007; Zhang et al., 2008) and with Rho family GTPases (He & Guo, 2009). The actin cytoskeleton that is essential for the delivery of the vesicle-associated subunits is not involved in the positioning of SEC3 (Finger et al., 1998; Wiederkehr et al., 2003; Hutagalung et al., 2009).
Relative to budding yeast, the role of the exocyst in plant cells remains unclear, although it could serve as a key component for the regulation of polarized exocytosis during cell expansion. All exocyst subunits are conserved in plants, and a plant-specific amplification of EXO70 genes has occurred during evolution, resulting in 23 EXO70 genes in Arabidopsis and 39 in rice (Eliás et al., 2003; Synek et al., 2006; Li et al., 2010). The expression patterns of these EXO70 genes are different, but they are all expressed in cells that are actively dividing or expanding (Li et al., 2010), supporting the idea that the exocyst is involved in the regulation of polarized exocytosis in plant cells.
Mutations in plant exocyst subunits that are encoded by one or two genes (SEC5, SEC6, SEC8, SEC15) cause defects in pollen germination and tube growth (Cole et al., 2005; Synek et al., 2006; Hála et al., 2008), suggesting an important function of the exocyst during plant cell tip growth. Mutations in the exocyst subunits that are encoded by more than two copies of genes (EXO84 (three genes) and EXO70 (23 genes)) cause growth defects of different degrees (Synek et al., 2006; Samuel et al., 2009; Fendrych et al., 2010; Kulich et al., 2010; Pecenková et al., 2011). Plant homologues of SEC3 have only been studied in maize. A mutation in the SEC3 encoding gene ROOTHAIRLESS1 results in the failure of correct root hair elongation (Wen et al., 2005). However, the expression patterns of maize SEC3 genes are not known, which makes it difficult to interpret this result. In Arabidopsis, SEC3 is encoded by two nearly identical genes in a tandem arrangement (Eliás et al., 2003). This has hampered the genetic analysis of SEC3 function in Arabidopsis. Interestingly, in Arabidopsis, the ICR1 (interactor of constitutive active ROP1) adaptor protein interacts with SEC3, which provides a putative link to activated ROP (Rho of plants) GTPases (Lavy et al., 2007). As ROP GTPases serve as intracellular polarity markers (Yang, 2008), this link suggests that exocyst recruitment could be (partially) ROP mediated.
Hála et al. (2008) have shown by immunocytochemistry that SEC6, SEC8 and EXO70A1 are enriched in the apex of growing tobacco pollen tubes, which is consistent with a role in polarized exocytosis. In tobacco Bright Yellow 2 (BY-2) suspension cultured cells, transient expression of Arabidopsis SEC5A, SEC15A, SEC15B and EXO84B fused to green fluorescent protein (GFP) resulted in fluorescent, globular structures in the perinuclear cytosol (Chong et al., 2010), whereas immunofluorescence revealed that the intracellular localization of EXO70 proteins differs depending on the isoform; they show either a cytoplasmic organization or localize to smaller or larger compartments with different degrees of co-localization with snares specific for early endosomes, late endosomes and the trans Golgi network (Chong et al., 2010; Wang et al., 2010). Distinct structures were also observed by Samuel et al. (2009) in stigma cells expressing red fluorescent protein (RFP):EXO70A1. On opening of the flowers, the RFP:EXO70A1 fluorescence relocated to the cell cortex, suggesting that EXO70A1 localization depends on the developmental stage. During cytokinesis, the Arabidopsis exocyst subunits SEC6, SEC8, SEC15B, EXO70A1 and EXO84B localize to the early cell plate, whereafter their localization on the cell plate disappears (Fendrych et al., 2010). Exocyst subunits reappear on the division wall for a short period after completion of cytokinesis (Fendrych et al., 2010).
Recent research has revealed that the exocyst subunits EXO84B, EXO70A1, SEC6 and SEC8 form distinct foci at the plasma membrane, with lifetimes ranging from 9.3 (EXO70A1) to 13.3 s (Fendrych et al., 2013). Co-localization studies have revealed that both EXO84B and SEC6 are present in 37% of these foci (Fendrych et al., 2013). As this study was carried out by variable-angle epifluorescence microscopy, only the plasma membrane localization was studied, and it was not investigated whether these exocyst subunits also localize to vesicles. Wang et al. (2010) showed that EXO70E2 localizes to discrete punctate structures at the plasma membrane and in the cytosol of Arabidopsis cells and tobacco BY-2 suspension cultured cells. The compartments at the plasma membrane are contained by two membranes, both of which are EXO70E2 decorated, and secretory. On secretion, the inner membrane is expelled into the apoplast. These organelles, which do not co-localize with any known organelle, have been named EXPO (exocyst-positive organelles; Wang et al., 2010). For a better understanding of the localization of different exocyst subunits and their assembly into multicomponent complexes, additional work is needed.
Here, we report the characterization of the SEC3A gene in Arabidopsis. Disruption of the SEC3A gene is embryo lethal, with defects in the acquisition of embryo polarity. During cytokinesis, SEC3A-GFP localizes to the early cell plate and to the completed division wall, respectively, similar to other exocyst subunits (Fendrych et al., 2010). In interphase cells, SEC3A-GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctate structures with discrete lifetimes. In tip growing root hairs, the puncta localize over the whole cell surface, and the amount of puncta does not decrease strongly in fully grown cells. Inhibition of exocytosis causes a strong reduction in the density of the puncta. As the density of SEC3A-GFP puncta does not depend on (the location of) cell expansion, this indicates that SEC3A mediates a type of exocytosis not related to cell growth. Our data show that the polar localization of SEC3A-GFP in the cell cortex occurs only during and just after cytokinesis, and that SEC3A-GFP puncta are evenly spread throughout the cell cortex in interphase cells.
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Our results can help to decipher the role of the exocyst in general and, in particular, of SEC3A in plant development. In interphase cells, SEC3A-GFP has a cytoplasmic localization and accumulates as immobile puncta at the plasma membrane with average lifetimes of 6.3–12.6 s depending on the cell type. Although SEC3A may be involved in exocytosis, it is not involved in the insertion of CESAs into the plasma membrane, and the density of SECA-GFP puncta does not depend on (polar) cell growth in interphase cells. SEC3A-GFP localizes to the cell plate during cytokinesis, but the lack of polarity in embryos of sec3a mutants suggests that SEC3A performs a function beyond the formation and/or correct positioning of cell plates in obtaining polarity during embryo development.
Interactions between different Arabidopsis exocyst subunits have been identified using different techniques. Hála et al. (2008) used chromatographic fractionation and yeast two-hybrid assays, whereas Pecenková et al. (2011) used yeast two-hybrid assays and fluorescence resonance energy transfer (FRET) analysis between several subunits. Together, these results strongly suggest that exocyst composition, and probably also function, is conserved in plants. Although co-fractionation shows that different exocyst subunits are in the same complex in plants (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15 and EXO70A1; Hála et al., 2008), the pairwise interactions found by the yeast two-hybrid assay provide additional insight into exocyst assembly. We have identified additional interactions between different exocyst subunits: SEC3A interacts with SEC5A, SEC15B interacts with EXO84C, EXO70A1 interacts with EXO84C, and EXO70H7 interacts with EXO84B. In addition, we determined two weak interactions in the yeast two-hybrid assay: SEC5A with SEC6, and SEC5A with EXO84C. Knowledge about interactions within the exocyst could aid in the deciphering of the exocyst structure.
SEC3A in cytokinesis
sec3a null mutants are embryonically lethal, which differs from defects in other exocyst subunit mutants that have been described (Cole et al., 2005; Synek et al., 2006; Samuel et al., 2009; Kulich et al., 2010; Pecenková et al., 2011). The successful transmission of the sec3a allele from the male and female gametophytes to the progeny allows us to study exocyst functioning in somatic development, which is not possible in exocyst mutants that are gametophytically lethal. The failure of the sec3a mutant embryo to develop from globular to heart shape shows that embryo polarization is not established correctly in the sec3a mutant. Although exocyst subunits localize to the cell plate (Fendrych et al., 2010; our results), the defective sec3a embryos show that cell plate formation is not disrupted. It is also unlikely that the exocyst plays a role in determining the orientation of cell divisions, as defects during embryo development only arise after the initial divisions that occur normally. Moreover, in the fass mutant, in which the orientation of cell division and cell expansion is disrupted, this does not interfere with embryonic pattern formation and cell polarity (Torres-Ruiz & Jürgens, 1994). The sec3a mutant phenotype resembles that of the gnom mutant, with the difference that embryo development in the sec3a mutant arrests earlier than in the gnom mutant (Mayer et al., 1991). As the complete lack of polarity in the gnom mutant is caused by defects in PIN cycling during polar auxin transport (Geldner et al., 2003), it is possible that SEC3A plays a role in polar auxin transport. As the non-lethal exocyst mutants, exo70A1 and sec8, display defects in PIN cycling (Drdová et al., 2013), SEC3A may function in PIN localization during embryo development. In addition, SEC3A interacts with ICR1, which could transduce ROP-mediated signaling to the exocyst (Lavy et al., 2007; Bloch et al., 2008); the exocyst could function as a ROP effector. ROP signaling has been implicated in many developmental processes, ranging from cell morphogenesis and differentiation to polar positioning of PIN proteins and the polarization of plant cell divisions (Yang & Fu, 2007; Humphries et al., 2011; Nagawa et al., 2012).
Localization and behavior of SEC3A-GFP puncta at the plasma membrane of interphase cells
SEC3A is cytoplasmic and localizes transiently to the plasma membrane in puncta, with an average lifetime varying from 6.3 to 12.6 s. There is no difference in the density of puncta between the growing root hair tip, the non-growing tube and the fully grown hair, and no difference between expanding and fully grown root epidermal cells. Thus, it is unlikely that SEC3A has a role in the exocytosis of cell wall matrix polysaccharides, unless the SEC3A-GFP puncta serve as an anchoring location for the remainder of the exocyst. In this case, although evenly spaced, SEC3A-GFP puncta in the plasma membrane may only mediate exocytotic events when interaction with the exocyst subunits on the exocytotic vesicle occurs. SEC3A could be compared with a door handle, always present, which requires an actor to open the door. This is unlikely as the other exocyst subunits, SEC6, SEC8, EXO70A1 and EXO84B, have a similar localization pattern (Fendrych et al., 2013). In addition, the non-polar localization of SEC3A differs from the situation in budding yeast, where SEC3 localizes to plasma membrane areas in which polarized exocytosis occurs, for example presumptive bud sites, the tips of budding cells and the mother–daughter cell neck during cytokinesis, where it plays a role in tethering secretory vesicles (Finger et al., 1998). In addition, in growing tobacco pollen tubes, Hála et al. (2008) showed the accumulation of SEC6, SEC8 and EXO70A1 in the apical region where expansion occurs. Unlike the polar localization of SEC3 in yeast and the polar localization of exocyst subunits in tip growing pollen tubes, SEC3A-GFP does not show polarized localization on the plasma membrane of polarly expanding plant cells.
EXO70E2 localizes to discrete punctate structures that are present both in association with the plasma membrane and in the cytosol. This is different from the localization of SEC3A-GFP which does not localize to punctate structures in the cytoplasm. The EXO70E2-containing compartments in the cytoplasm were termed EXPO, as they did not co-localize with any conventional organelle (Wang et al., 2010). Unlike EXO70E2, SEC3A-GFP only localizes to discrete puncta at the plasma membrane, and not in the cytoplasm. In budding yeast, SEC3A functions as a landmark protein at the plasma membrane, where most other exocyst subunits (SEC5, SEC6, SEC8, SEC10, SEC15 and EXO84) are recruited via attachment to exocytotic vesicles (Zhang et al., 2008; Hutagalung et al., 2009). Budding yeast EXO70 localizes to the plasma membrane but, unlike SEC3, its localization is dependent on the actin cytoskeleton (Hutagalung et al., 2009). This is consistent with the localization of EXO70A1 to the plasma membrane (Fendrych et al., 2013) and EXO70E2 to both EXPOs and plasma membrane puncta in plant cells.
Plasma membrane-localized SEC3A-GFP puncta have discrete lifetimes. The fluorescence intensity during the lifetime of a punctum can be divided into three phases: first, it gradually increases; it then remains constant for a short time span and, finally, it decreases gradually. This suggests the recruitment of multiple SEC3A proteins over time from the cytosol, followed by a gradual dissociation of SEC3A proteins in the final phase. The yeast exocyst is thought to consist of single or, at most, a few proteins per subunit (Munson & Novick, 2006). As the puncta in the plasma membrane of interphase cells are unlikely to represent single SEC3A-GFP proteins, our results show that the plant exocyst either consists of multiple SEC3A proteins or that multiple exocyst-mediated events occur in one punctum. The decrease in density of SEC3A-GFP puncta during BFA treatment suggests that the presence of SEC3A at the plasma membrane is dependent on the presence of Golgi-derived vesicles or vesicle-associated factors, for example other exocyst subunits. Interestingly, the lifetime of SEC3A-GFP puncta is shorter than that of the other exocyst subunits that display this localization. In comparable cell types, elongating root epidermal cells, the lifetime of SEC3A-GFP puncta (6.7 ± 3.6 s) is almost 3 s shorter than that of GFP-EXO70A1 (9.3 s) and almost 7 s shorter than that of GFP-SEC8 (13.3 s; Fendrych et al., 2013). The short SEC3A lifetime compared with that of other exocyst subunits suggests that it is unlikely that SEC3A is recruited to the plasma membrane before the other exocyst subunits, and draws its role as a landmark protein into question. To be conclusive, thorough co-localization studies between SEC3A and the other exocyst subunits should be performed.
As discussed above, the similar, uniform density and similar lifetimes of SEC3A-GFP puncta at the plasma membrane suggest that SEC3A mediates an exocytotic event that is not related to (polarized) cell expansion. The only type of exocytosis that occurs over the cell surface, which can currently be detected using fluorescence microscopy, is that of CESA into the plasma membrane (Crowell et al., 2009; Gutierrez et al., 2009). We did not find co-localization between SEC3A and CESA insertion into the plasma membrane. Therefore, SEC3A does not appear to be involved in the insertion of CESA, but may mediate exocytosis of the cell wall matrix via Golgi vesicles. However, we have not been able to show this directly and cannot exclude the possibility that SEC3A mediates exocytotic events not related to cell wall formation, for example the insertion of trans-membrane receptors or ion channels into the plasma membrane. Pecenková et al. (2011) have shown the involvement of Exo70B2 and Exo70H1 in plant defence against pathogens. The insertion of receptors into the plasma membrane by an exocytotic mechanism during pathogen defence could be mediated by the SEC3A-GFP puncta.