ABNORMAL POLLEN TUBE GUIDANCE1, an Endoplasmic Reticulum-Localized Mannosyltransferase Homolog of GLYCOSYLPHOSPHATIDYLINOSITOL10 in Yeast and PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B in Human,Is Required for Arabidopsis Pollen Tube Micropylar Guidance and Embryo Development

The perception and response of pollen tubes to the female guidance signals are crucial for directional pollen tube growth inside female tissues, which leads to successful reproduction. In pursuing the mechanisms underlying this biological process, we identified the Arabidopsis (Arabidopsis thaliana) abnormal pollen tube guidance1 (aptg1) mutant, whose pollen tubes showed compromised micropylar guidance. In addition to its male defect, the aptg1 mutant showed embryo lethality. APTG1 encodes a putative mannosyltransferase homolog to human PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS B and yeast (Saccharomyces cerevisiae) GLYCOSYLPHOSPHATIDYLINOSITOL10 (GPI10), both of which are involved in the biosynthesis of GPI anchors. We found that APTG1 was expressed in most plant tissues, including mature pollen, pollen tubes, mature embryo sacs, and developing embryos. By fluorescence colabeling, we showed that APTG1 was localized in the endoplasmic reticulum, where GPI anchors are synthesized. Disruption of APTG1 affected the localization of COBRA-LIKE10, a GPI-anchored protein important for pollen tube growth and guidance. The results shown here demonstrate that APTG1 is involved in both vegetative and reproductive development in Arabidopsis, likely through processing and proper targeting of GPI-anchored proteins.Double fertilization is the biological basis for seed propagation and plant reproduction in angiosperms. Pollen tubes grow through maternal tissue to deliver the immobile sperm cells into the female gametophyte (embryo sac). During this process, pollen tube guidance into the micropyle is a critical step and is precisely regulated (Dresselhaus and Franklin-Tong, 2013). Female guidance signals are generated by both sporophytic and gametophytic tissues and operate at different stages during pollen tube growth. The sporophytic signal directs the growth of pollen tubes in the stigma, style, and transmitting tract. The signal that induces pollen tubes to turn to the funiculus and grow into the micropyle is termed gametophytic guidance (Shimizu and Okada, 2000; Higashiyama et al., 2003). Extensive cellular and genetic studies have demonstrated that female gametophytes play key roles in the micropylar guidance of pollen tubes (Kasahara et al., 2005; Márton et al., 2005; Chen et al., 2007; Alandete-Saez et al., 2008; Okuda et al., 2009; Kessler and Grossniklaus, 2011; Takeuchi and Higashiyama, 2011). The molecular natures of such guidance signals have been gradually revealed in recent years (i.e. small peptides secreted by the female gametophyte, egg apparatus, or synergid cells; Márton et al., 2005; Jones-Rhoades et al., 2007; Okuda et al., 2009).Pollen tubes need to perceive the female guidance signals at the cell surface to initiate intracellular responses for directional growth. However, the mechanisms of pollen tube perception are still obscure. A few male factors involved in signal perception during pollen tube growth into ovules have been identified. For example, the Arabidopsis (Arabidopsis thaliana) sperm cell-specific protein HAPLESS2/GENERATIVE CELL-SPECIFIC1 was necessary for pollen tubes to target the micropyle (von Besser et al., 2006). Arabidopsis CATION/PROTON EXCHANGER21 (CHX21) and CHX23 encode K⁺ transporters in growing pollen tubes. Pollen grains of the chx21 chx23 double mutant germinated and extended a normal tube in the transmitting tract, but their targeting of the funiculus failed (Lu et al., 2011). Arabidopsis POLLEN DEFECTIVE IN GUIDANCE1 (POD1) was expressed in pollen grains, pollen tubes, and synergid cells. The pod1 pollen tubes showed defective micropylar guidance (Li et al., 2011). The tip of the pollen tube has been hypothesized to be the site of cue perception for micropyle-directed growth. The Arabidopsis Rab GTPase RABA4D was localized at the tips of growing pollen tubes. Pollen tubes with defective RABA4D had severely reduced growth rates and ovule targeting (Szumlanski and Nielsen, 2009). Recently, two receptor-like kinases at the apical plasma membrane (PM) of growing pollen tubes, LOST IN POLLEN TUBE GUIDANCE1 (LIP1) and LIP2, were demonstrated to guide pollen tubes to the micropyle by perceiving the AtLURE1 signal from synergid cells (Liu et al., 2013).Glycosylphosphatidylinositol (GPI) anchoring provides a strategy for targeting proteins to the outer layer of the PMs in eukaryotic cells. GPI anchors are synthesized inside the endoplasmic reticulum (ER) and are attached to proteins by posttranslational modifications in the ER. After processing, GPI-anchored proteins (GPI-APs) are transported to the cell surface following an unknown trafficking route and anchored at the cell surface (Maeda and Kinoshita, 2011). GPI-APs play very important roles in plant reproductive development (Gillmor et al., 2005; Ching et al., 2006; DeBono et al., 2009). An Arabidopsis putative GPI-AP, LORELEI, functioned in pollen tube reception of female signals, double fertilization, and early seed development (Capron et al., 2008; Tsukamoto et al., 2010). Arabidopsis COBRA-LIKE10 (COBL10), a GPI-AP, regulates the polar deposition of wall components in pollen tubes growing inside female tissues and is critical for micropylar guidance (Li et al., 2013). The conserved backbone of GPI anchors in eukaryotes is ethanolamine phosphate-6-Man-α-1,2-Man-α-1,6-Man-α-1,4-glucosamine-α-1,6-myoinositol phospholipid. During the biosynthesis of GPI anchors, monosaccharides, fatty acids, and phosphoethanolamines are sequentially added onto phosphatidylinositol. This process involves at least 16 enzymes and cofactors in mammals, including PHOSPHATIDYLINOSITOL GLYCAN ANCHOR BIOSYNTHESIS (PIG) A, B, C, F, G, H, L, M, N, O, P, Q, V, W, X, and Y (Maeda and Kinoshita, 2011). The core structure of the GPI anchor contains three Man residues donated by the substrate dolichol-phosphate-Man. GPI mannosyltransferases were required for adding the three Man residues of the GPI anchor in the ER lumen (Maeda and Kinoshita, 2011). Arabidopsis PEANUT1 (PNT1) is a homolog of the mammalian GPI mannosyltransferase PIG-M, involved in the addition of the first Man during the biosynthesis of the GPI anchor. The pnt1 mutant showed the defect of pollen viability and embryo development (Gillmor et al., 2005). PIG-B of human and GPI10 of yeast (Saccharomyces cerevisiae) encode GLYCOSYLPHOSPHATIDYLINOSITOL MANNOSYLTRANSFERASE3, involved in the addition of the third Man during the biosynthesis of the GPI anchor (Takahashi et al., 1996; Sütterlin et al., 1998). Mutation of PIG-B and GPI10 resulted in the accumulation of the GPI intermediate Man2-glucosamine-(acyl) phosphatidylinositol and led to cell death in yeast.In this study, we identified the ER-localized ABNORMAL POLLEN TUBE GUIDANCE1 (APTG1), an Arabidopsis homolog of PIG-B and GPI10. Pollen tubes of the aptg1 mutant showed compromised directional growth to the micropyle and lost the apical PM localization of COBL10. Besides the male defect, the mutant showed embryo lethality. In addition, reducing the expression of APTG1 resulted in defective seedling growth, indicating that APTG1 plays important roles in both reproductive and vegetative development.     

A Calcium Sensor-Regulated Protein Kinase,CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19, Is Required for Pollen Tube Growth and Polarity

Calcium plays an essential role in pollen tube tip growth. However, little is known concerning the molecular basis of the signaling pathways involved. Here, we identified Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (CIPK19) as an important element to pollen tube growth through a functional survey for CIPK family members. The CIPK19 gene was specifically expressed in pollen grains and pollen tubes, and its overexpression induced severe loss of polarity in pollen tube growth. In the CIPK19 loss-of-function mutant, tube growth and polarity were significantly impaired, as demonstrated by both in vitro and in vivo pollen tube growth assays. Genetic analysis indicated that disruption of CIPK19 resulted in a male-specific transmission defect. Furthermore, loss of polarity induced by CIPK19 overexpression was associated with elevated cytosolic Ca²⁺ throughout the bulging tip, whereas LaCl₃, a Ca²⁺ influx blocker, rescued CIPK19 overexpression-induced growth inhibition. Our results suggest that CIPK19 may be involved in maintaining Ca²⁺ homeostasis through its potential function in the modulation of Ca²⁺ influx.In flowering plants, fertilization is mediated by pollen tubes that extend directionally toward the ovule for sperm delivery (Krichevsky et al., 2007; Johnson, 2012). The formation of these elongated tubular structures is dependent on extreme polar growth (termed tip growth), in which cell expansion occurs exclusively in the very apical area (Yang, 2008; Rounds and Bezanilla, 2013). As this type of tip growth is amenable to genetic manipulation and cell biological analysis, the pollen tube is an excellent model system for the functional analysis of essential genes involved in polarity control and fertilization (Yang, 2008; Qin and Yang, 2011; Bloch and Yalovsky, 2013).It is well established that Ca²⁺ plays a critical role in pollen germination and tube growth (Konrad et al., 2011; Hepler et al., 2012). A steep tip-focused Ca²⁺ gradient has been detected at the tip of elongating pollen tubes (Rathore et al., 1991; Pierson et al., 1994; Hepler, 1997). In previous studies, artificial dissipation of the Ca²⁺ gradient seriously inhibited tip growth of pollen tubes, whereas elevation of internal Ca²⁺ level induced bending of the growth axis toward the zone of higher Ca²⁺. These studies suggest that Ca²⁺ not only controls pollen tube elongation but also modulates growth orientation (Miller et al., 1992; Malho et al., 1994; Malho and Trewavas, 1996; Hepler, 1997). These Ca²⁺ signatures are perceived and relayed to downstream responses by a complex toolkit of Ca²⁺-binding proteins that function as Ca²⁺ sensors (Yang and Poovaiah, 2003; Harper et al., 2004; Dodd et al., 2010).To date, four major Ca²⁺ sensor families have been identified in Arabidopsis (Arabidopsis thaliana), including calcium-dependent protein kinase, calmodulin (CaM), calmodulin-like (CML), and CALCINEURIN B-LIKE (CBL) proteins (Luan et al., 2002, 2009; Yang and Poovaiah, 2003; Harper et al., 2004). Calcium-dependent protein kinase family members comprise a kinase domain and a CaM-like domain in a single protein; thus, they act not only as a Ca²⁺ sensor but also as an effector, designated as sensor responders (Cheng et al., 2002). In contrast, CaM, CML, and CBL proteins do not have any enzymatic domains but transmit Ca²⁺ signals to downstream targets via Ca²⁺-dependent protein-protein interactions. Therefore, they have been designated as sensor relays (McCormack et al., 2005). While CaM and CML proteins interact with a diverse array of target proteins, it is generally accepted that CBLs interact specifically with a group of Ser/Thr protein kinases termed CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASEs (CIPKs; Luan et al., 2002; Kolukisaoglu et al., 2004).In Arabidopsis, several CBLs coupled with their target CIPKs have been demonstrated to function in the regulation of ion homeostasis and stress responses (Luan et al., 2009). Under salt stress, SALT OVERLY SENSITIVE3 (SOS3)/CBL4-SOS2/CIPK24 regulate SOS1 at the plasma membrane for Na⁺ exclusion, whereas CBL10-CIPK24 complexes appear to regulate Na⁺ sequestration at the tonoplast (Liu et al., 2000; Qiu et al., 2002; Kim et al., 2007; Quan et al., 2007). For low-K⁺ stress, CBL1 and CBL9, with 87% amino acid sequence identity, interact with CIPK23, which regulates a voltage-gated ion channel (ARABIDOPSIS K⁺ TRANSPORTER1) to mediate the uptake of K⁺ in root hairs (Li et al., 2006; Xu et al., 2006; Cheong et al., 2007). In addition, CBL1 integrates plant responses to cold, drought, salinity, and hyperosmotic stresses (Albrecht et al., 2003; Cheong et al., 2003), and CBL9 is involved in abscisic acid signaling and biosynthesis during seed germination (Pandey et al., 2004). Over the past decade, the functions of CBL-CIPK complexes in abiotic stress tolerance have been studied extensively, but only limited studies focus on CBL family members in pollen tube growth. For example, CBL3 overexpression caused a defective phenotype in pollen tube growth (Zhou et al., 2009). Overexpression of CBL1 or its closest homolog CBL9 inhibited pollen germination and perturbed tube growth at high external K⁺, whereas disruption of CBL1 and CBL9 leads to a significantly reduced growth rate of pollen tubes under low-K⁺ conditions (Mähs et al., 2013). The potential roles of CIPKs in pollen tubes so far appear to be completely unknown.In this study, we demonstrated that Arabidopsis CIPK19, a CIPK specifically expressed in pollen grains and pollen tubes, functions in pollen tube tip growth, providing a new insight into the function of the CBL-CIPK network in the control of growth polarity during pollen tube extension in fertilization.     

The Apical Actin Fringe Contributes to Localized Cell Wall Deposition and Polarized Growth in the Lily Pollen Tube

In lily (Lilium formosanum) pollen tubes, pectin, a major component of the cell wall, is delivered through regulated exocytosis. The targeted transport and secretion of the pectin-containing vesicles may be controlled by the cortical actin fringe at the pollen tube apex. Here, we address the role of the actin fringe using three different inhibitors of growth: brefeldin A, latrunculin B, and potassium cyanide. Brefeldin A blocks membrane trafficking and inhibits exocytosis in pollen tubes; it also leads to the degradation of the actin fringe and the formation of an aggregate of filamentous actin at the base of the clear zone. Latrunculin B, which depolymerizes filamentous actin, markedly slows growth but allows focused pectin deposition to continue. Of note, the locus of deposition shifts frequently and correlates with changes in the direction of growth. Finally, potassium cyanide, an electron transport chain inhibitor, briefly stops growth while causing the actin fringe to completely disappear. Pectin deposition continues but lacks focus, instead being delivered in a wide arc across the pollen tube tip. These data support a model in which the actin fringe contributes to the focused secretion of pectin to the apical cell wall and, thus, to the polarized growth of the pollen tube.Pollen tubes provide an excellent model for studying the molecular and physiological processes that lead to polarized cell growth. Because all plant cell growth results from the regulated yielding of the cell wall in response to uniform turgor pressure (Winship et al., 2010; Rojas et al., 2011), the cell wall of the pollen tube must yield only at a particular spot: the cell apex, or tip. To accomplish the extraordinary growth rates seen in many species, and to balance the thinning of the apical wall due to rapid expansion, the pollen tube delivers prodigious amounts of wall material, largely methoxylated pectins, to the tip in a coordinated manner. Recent studies suggest that the targeted exocytosis increases the extensibility of the cell wall matrix at the tip, which then yields to the existing turgor pressure, permitting the tip to extend or grow (McKenna et al., 2009; Hepler et al., 2013). There are many factors that influence exocytosis in growing pollen tubes; in this study, we investigate the role of the apical actin fringe.For many years, it has been known that an actin structure exists near the pollen tube tip, yet its exact form has been a matter of some contention (Kost et al., 1998; Lovy-Wheeler et al., 2005; Wilsen et al., 2006; Cheung et al., 2008; Vidali et al., 2009; Qu et al., 2013). The apical actin structure has been variously described as a fringe, a basket, a collar, or a mesh. Using rapid freeze fixation of lily (Lilium formosanum) pollen tubes followed by staining with anti-actin antibodies, the structure appears as a dense fringe of longitudinally oriented microfilaments, beginning 1 to 5 µm behind the apex and extending 5 to 10 µm basally. The actin filaments are positioned in the cortical cytoplasm close to the plasma membrane (Lovy-Wheeler et al., 2005). More recently, we used Lifeact-mEGFP, a probe that consistently labels this palisade of longitudinally oriented microfilaments in living cells (Vidali et al., 2009; Fig. 1A, left column). For the purposes of this study, we will refer to this apical organization of actin as a fringe.Open in a separate windowFigure 1.The actin fringe and the thickened pollen tube tip wall are stable, although dynamic, structures during pollen tube growth. A, The left column shows a pollen tube transformed with Lifeact-mEGFP imaged with a spinning-disc confocal microscope. Maximal projections from every 15 s are shown. The right column shows epifluorescence images of a pollen tube stained with PI. Again, images captured every 15 s are shown. Bars = 10 μm. B, The data from the pollen tube in A expressing Lifeact-mEGFP were subjected to kymograph analysis using an 11-pixel strip along the image’s midline. C, The first three frames from the pollen tube in A and B were assigned the colors red, blue, and green, respectively, and then overlaid. Areas with white show the overlap of all three. The fringe is stable, but most of its constituent actin is not shared between frames.Many lines of evidence demonstrate that actin is required for pollen tube growth. Latrunculin B (LatB), which blocks actin polymerization, inhibits pollen tube growth and disrupts the cortical fringe at concentrations as low as 2 nm. Higher concentrations are needed to block pollen grain germination and cytoplasmic streaming (Gibbon et al., 1999; Vidali et al., 2001). Actin-binding proteins, including actin depolymerizing factor-cofilin, formin, profilin, and villin, and signaling proteins, such as Rho-of-Plants (ROP) GTPases and their effectors (ROP interacting crib-containing proteins [RICs]), also have been shown to play critical roles in growth and actin dynamics (Fu et al., 2001; Vidali et al., 2001; Allwood et al., 2002; Chen et al., 2002; Cheung and Wu, 2004; McKenna et al., 2004; Gu et al., 2005; Ye et al., 2009; Cheung et al., 2010; Staiger et al., 2010; Zhang et al., 2010a; Qu et al., 2013; van Gisbergen and Bezanilla, 2013).Our understanding of the process of exocytosis and pollen tube elongation has been influenced by ultrastructural images of pollen tube tips, which reveal an apical zone dense with vesicles (Cresti et al., 1987; Heslop-Harrison, 1987; Lancelle et al., 1987; Steer and Steer, 1989; Lancelle and Hepler, 1992; Derksen et al., 1995). It has long been assumed that these represent exocytotic vesicles destined to deliver new cell wall material. This model of polarized secretion has been challenged in recent years in studies using FM dyes. Two groups have suggested that exocytosis occurs in a circumpolar annular zone (Bove et al., 2008; Zonia and Munnik, 2008). However, other studies, using fluorescent beads attached to the cell surface, indicate that the maximal rate of expansion, and of necessity the greatest deposition of cell wall material, occurs at the apex along the polar axis of the tube (Dumais et al., 2006; Rojas et al., 2011). Similarly, our experiments with propidium iodide (PI; McKenna et al., 2009; Rounds et al., 2011a) and pectin methyl esterase fused to GFP (McKenna et al., 2009) show that the wall is thickest at the very tip and suggest that wall materials are deposited at the polar axis, consistent with the initial model of exocytosis (Lancelle and Hepler, 1992). Experiments using tobacco (Nicotiana tabacum) pollen and a receptor-like kinase fused to GFP also indicate that exocytosis occurs largely at the apical polar axis (Lee et al., 2008).Many researchers argue that apical actin is critical for exocytosis (Lee et al., 2008; Cheung et al., 2010; Qin and Yang, 2011; Yan and Yang, 2012). More specifically, recent work suggests that the fringe participates in targeting vesicles and thereby contributes to changes in growth direction (Kroeger et al., 2009; Bou Daher and Geitmann, 2011; Dong et al., 2012). In this article, using three different inhibitors, namely brefeldin A (BFA), LatB, and potassium cyanide (KCN), we test the hypothesis that polarized pectin deposition in pollen tubes requires the actin fringe. Our data show that during normal growth, pectin deposition is focused to the apex along the polar axis of the tube. However, when growth is modulated, different end points arise, depending on the inhibitor. With BFA, exocytosis stops completely, and the fringe disappears, with the appearance of an actin aggregate at the base of the clear zone. LatB, as shown previously (Vidali et al., 2009), incompletely degrades the actin fringe and leaves a rim of F-actin around the apical dome. Here, we show that, in the presence of LatB, pectin deposition continues, with the focus of this activity shifting in position frequently as the slowly elongating pollen tube changes direction. With KCN, the actin fringe degrades completely, but exocytosis continues and becomes depolarized, with pectin deposits now occurring across a wide arc of the apical dome. This dome often swells as deposition continues, only stopping once normal growth resumes. Taken together, these results support a role for the actin fringe in controlling the polarity of growth in the lily pollen tube.     

Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth

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 green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine 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 subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-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 the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.Pollen tube growth provides a unique model system for studying the role of exocytosis in cell morphogenesis. Pollen tubes are characterized by a highly rapid polarized unidirectional tip growth. Given the relative simplicity of their structure, fast growth rates, haploid genome content, and ability to grow under in vitro culture conditions, pollen tubes provide an extremely attractive system for studying cell morphogenesis. Furthermore, the growth characteristics of pollen tubes resemble those of root hairs, moss protonema, and fungal hyphae and to some extent can be paralleled to neurite growth (Chebli and Geitmann, 2007; Cheung and Wu, 2008; Guan et al., 2013; Hepler and Winship, 2015).It is well established that oscillating polarized exocytosis is fundamental for pollen tube development and determines growth rate (Bove et al., 2008; McKenna et al., 2009; Chebli et al., 2013). Exocytosis is required for the delivery of membrane and cell wall components to the growing tip. Yet, the exact location where exocytosis takes place is under debate. Ultrastructural studies showing the accumulation of vesicles at the tip suggested that exocytosis takes place at the tip (Lancelle et al., 1987; Lancelle and Hepler, 1992; Derksen et al., 1995), which was further supported by studies on the dynamics of cell wall thickness (Rojas et al., 2011), secretion of pectin methyl esterase (PME) and PME inhibitor, and staining of pectin by propidium iodide (PI; Röckel et al., 2008; Rounds et al., 2014). Conversely, based on colabeling with FM1-43 and FM4-64, it was concluded that exocytosis takes place in a subapical collar located in the transition zone between the tip and the shank, as well as at the shank, but not at the tip (Bove et al., 2008; Zonia and Munnik, 2008). In agreement, the pollen tube-specific syntaxin GFP-SYP124 was observed in the inverted cone, 10 to 25 μm away from the tip (Silva et al., 2010), and fluorescence recovery after photobleaching experiments with FM dyes also have indicated that exocytosis takes place at the subapical region (Bove et al., 2008; Moscatelli et al., 2012; Idilli et al., 2013). Yet, based on pollen tube reorientation experiments in a microfluidics device, it was concluded that growth takes place at the tip rather than at a subapical collar located in the transition zone between the apex and the shank (Sanati Nezhad et al., 2014). The tip-based growth is in agreement with exocytosis taking place at the tip. Presumably, part of the disagreement regarding the site of exocytosis resulted from the lack of intracellular markers for exocytosis (Cheung and Wu, 2008; Hepler and Winship, 2015), and as a result, the relationship between the FM dye-labeled inverted cone and exocytotic events during pollen tube growth is not fully understood.In many cell types, the process of secretory vesicles tethering and docking prior to fusion with the plasma membrane is initially mediated by an evolutionarily conserved tethering complex known as the exocyst. The exocyst is a heterooligomeric protein complex composed of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Studies originally based on budding yeast (Saccharomyces cerevisiae) have shown that the exocyst functions as an effector of Rab and Rho small GTPases that specifies the sites of vesicle docking and fusion at the plasma membrane in both space and time (Guo et al., 2001; Zhang et al., 2001). Support for the function of the exocyst in vesicle tethering was demonstrated recently by ectopic Sec3p-dependent vesicle recruitment to the mitochondria (Luo et al., 2014).Land plants contain all subunits of the exocyst complex, which were shown to form the functional complex (Elias et al., 2003; Cole et al., 2005; Synek et al., 2006; Hála et al., 2008). Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al., 2005; Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010; Kulich et al., 2010; Pecenková et al., 2011; Safavian and Goring, 2013; Wu et al., 2013; Safavian et al., 2015; Zhang et al., 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al., 2005; Hála et al., 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al., 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al., 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al., 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013; Safavian et al., 2015) have implicated the exocyst in polarized exocytosis in plants. Given their function, it was likely that exocyst subunits could be used as markers for polarized exocytosis. Furthermore, it could also be hypothesized that, by studying the mechanisms that underlie the association of the exocyst complex with the plasma membrane, it should be possible to identify mechanisms underlying the regulation of polarized exocytosis (Guan et al., 2013). Moreover, since the interaction of exocytotic vesicles with the exocyst is transient and marks the site(s) of active exocytosis in the membrane, fluorescently labeled exocyst subunits could be used as markers for exocytosis while avoiding potential imaging artifacts stemming from pollen tube tips densely populated with vesicles.We have shown previously that the ROP effector ICR1 can interact with SEC3a and that ROPs can recruit SEC3a-ICR1 complexes to the plasma membrane (Lavy et al., 2007). However, ICR1 is not expressed in pollen tubes, suggesting that SEC3a membrane binding in these cells is likely dependent on other factors. In yeast, the interaction of Sec3p and Exo70p subunits with the plasma membrane is critical for exocyst function (He and Guo, 2009). It has been shown that the membrane binding of both Sec3p and Exo70p is facilitated by their interaction with phosphatidylinositol 4,5-bisphosphate (PIP₂; He et al., 2007; Zhang et al., 2008). The yeast Exo70p interacts with PIP₂ via a number of positively charged residues distributed along the protein, with the highest number located at the C-terminal end (Pleskot et al., 2015). It has been suggested that yeast Sec3p interacts with PIP₂ through N-terminal basic residues (Zhang et al., 2008). These data were further corroborated by x-ray crystallography studies, which showed that the yeast Sec3p N-terminal region forms a Pleckstrin homology (PH) domain fold (Baek et al., 2010; Yamashita et al., 2010), a PIP₂ interaction motif (Lemmon, 2008).The localization of the exocyst subunits has been addressed in several studies. In Arabidopsis root hairs and root epidermis cells, SEC3a-GFP was observed in puncta distributed throughout the cell (Zhang et al., 2013). Studies on the Arabidopsis EXO70 subunits EXO70E2, EXO70A1, and EXO70B1 revealed them to be localized in distinct compartments that were termed exocyst-positive organelles (Wang et al., 2010). The exocyst-positive organelles, visualized mostly by ectopic expression, were shown to be cytoplasmic double membrane organelles that can fuse with the plasma membrane and secrete their contents to the apoplast in an exosome-like manner. It is not yet known whether other exocyst subunits also are localized to the same organelles and what might be the biological function of this putative compartment (Wang et al., 2010; Lin et al., 2015). In differentiating xylem cells, two coiled-coil proteins termed VESICLE TETHERING1 and VESICLE TETHERING2 recruit EXO70A1-positive puncta to microtubules via the GOLGI COMPLEX2 protein (Oda et al., 2015). Importantly, the functionality of the XFP fusion proteins used for the localization studies described above was not tested, and in most cases, the fusion proteins were overexpressed. Therefore, the functional localization of the exocyst is still unclear.Here, we studied the function and subcellular localization of the Arabidopsis exocyst SEC3a subunit using a combination of genetics, cell biology, biochemistry, and structural modeling approaches. Our results show that SEC3a is essential for the determination of pollen tube tip germination site and growth. Partial complementation of sec3a resulted in the formation of pollen with multiple pollen tube tips. In Arabidopsis growing pollen tubes, SEC3a localization is dynamic, and it accumulates in domains of polarized secretion, at or close to the tip plasma membrane (PM). Labeling of GFP-SEC3-expressing pollen with FM4-64 revealed the spatial correlation between polarized exocytosis and endocytic recycling. Furthermore, the association of SEC3a with PM at the tip marks the direction of tube elongation and positively correlates with the deposition of PI-labeled pectins and specific anti-esterified pectin antibodies in the cell wall. In tobacco (Nicotiana tabacum), the mechanisms underlying SEC3a interaction with the PM and its subcellular distribution depend on pollen tube growth mode and involve the interaction with PIP2 through the N-terminal PH domain. Collectively, our results highlight the function of SEC3a as a polarity determinant that links between polarized exocytosis and cell morphogenesis. The correlation between exocyst function and distribution in pollen tubes provides an explanation for some of the current discrepancies regarding the localization of exocytosis.     

A Pollen Protein,NaPCCP, That Binds Pistil Arabinogalactan Proteins Also Binds Phosphatidylinositol 3-Phosphate and Associates with the Pollen Tube Endomembrane System

As pollen tubes grow toward the ovary, they are in constant contact with the pistil extracellular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions, NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, and live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCP∷green fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabinogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system.Angiosperm sexual reproduction requires pollen transfer to a receptive stigma followed by its hydration, germination, and pollen tube growth. Pollen tubes grow through the stigma and style toward the ovule, where the sperm cells are discharged for fertilization. Pollen tubes do not divide; rather, they extend through tip growth while periodically producing callose plugs, separating highly vacuolated distal regions from the actively growing tip (Taylor and Hepler, 1997). The tip region shows strong zonation. An apical region or clear zone, a subapical, organelle-rich zone, a nuclear zone, and a distal vacuolated zone or plug region that may extend several centimeters are easily recognized (Mascarenhas, 1993). Proper deposition of wall material and rapid tube extension require coordination between GTPase-regulated trafficking pathways, the cytoskeleton, signaling pathways, and oscillatory ion and water fluxes (Li et al., 1999; Fu et al., 2001; Zonia et al., 2002; Camacho and Malhó, 2003; Chen et al., 2003; de Graaf et al., 2005; Gu et al., 2005).Pollen tube endomembrane system dynamics are critical for growth: wall materials are deposited by exocytosis, and the membrane is recovered by endocytosis (Picton and Steer, 1983; Cheung and Wu, 2008). Exocytosis of material synthesized in the Golgi occurs near the tip (Cheung et al., 2002). Additional wall material is produced by membrane-bound callose synthase, but this occurs behind the tip (Brownfield et al., 2007). Distinct endocytosis zones have been identified by pulse-chase membrane labeling, observations of charged nanoparticles, and electron microscopy (Derksen et al., 1995; Moscatelli et al., 2007; Zonia and Munnik, 2008). Clathrin-independent endocytosis occurs at the pollen tube apex; endocytic vesicles clearly contribute to vesicle populations in the clear zone once thought to be composed entirely of exocytic vesicles (Moscatelli et al., 2007; Bove et al., 2008; Zonia and Munnik, 2008). Inhibitor studies suggest that clathrin-dependent endocytosis occurs in the organelle-rich zone a few micrometers back from the tip (Moscatelli et al., 2007). Furthermore, coated vesicles have been observed from 6 to 15 μm from the tip by electron microscopy (Derksen et al., 1995).Pollen-pistil interactions influence pollen tube growth either positively or negatively. Positive effects are evident from the observation that pollen tubes grow as much as 10 times faster and achieve much greater lengths in planta than in culture (Cheung et al., 2000). Self-incompatibility (SI) systems provide the best understood examples of negative effects. In SI, pollen-pistil interactions cause rejection of closely related pollen tubes (de Nettancourt, 2001).Arabinogalactan proteins (AGPs) secreted into the pistil extracellular matrix (ECM) play key roles in both positive and negative interactions, but the underlying molecular interactions with pollen tubes are just beginning to be understood. The transmitting tract-specific (TTS) glycoprotein (Cheung et al., 1995; Wu et al., 1995, 2000) and the 120-kD glycoprotein (120K; Hancock et al., 2005) are pistil AGPs implicated in pollination in Nicotiana. Both are abundant components of the pistil ECM (Cheung et al., 1995; Lind et al., 1996) and share a conserved Cys-rich C-terminal domain (CTD). TTS was first described in Nicotiana tabacum (i.e. NtTTS) as a pollen tube attractant. Pollen tubes grow toward TTS in culture, and its glycosylation levels progressively increase closer to the ovary (Cheung et al., 1995). Pollen tubes deglycosylate TTS, which suggests that TTS may act as a nutritive factor (Wu et al., 1995) and, thus, positively affect pollen tube growth.120K is implicated in SI in Nicotiana alata (Cruz-Garcia et al., 2005; Hancock et al., 2005), a species that displays S-RNase-based gametophytic SI (McClure et al., 1989). In SI, compatibility is controlled by the polymorphic S-locus; pollen is rejected if its S-haplotype matches either of the two S-haplotypes in the diploid pistil (de Nettancourt, 2001). Each S-haplotype is unique and encodes separate pollen- and pistil-specificity genes (Kao and Tsukamoto, 2004). S-RNases determine specificity on the pistil side and directly inhibit the growth of closely related pollen tubes (McClure et al., 1989). S-locus F-box proteins (SLF/SFB) control specificity on the pollen side (Sijacic et al., 2004). SLF/SFB proteins bind S-RNase in vitro and appear to form several distinct complexes with other pollen proteins (Qiao et al., 2004; Hua and Kao, 2006; Huang et al., 2006). SI, therefore, is a clear example of inhibitory pollen-pistil interactions: interaction between a pistil protein, S-RNase, and a pollen protein, SLF/SFB, determines compatibility. However, other pistil factors are also required for SI (McClure et al., 1999; Hancock et al., 2005; McClure and Franklin-Tong, 2006). 120K, for example, is required for SI but does not directly contribute to S-specificity (Hancock et al., 2005).120K was first identified as an abundant component of the transmitting tract ECM that contains both arabinogalactan and extensin-like carbohydrate moieties (Lind et al., 1994). 120K is an S-RNase-binding protein that is taken up by growing pollen tubes (Lind et al., 1996; Cruz-Garcia et al., 2005; Goldraij et al., 2006). Immunolocalization studies show 120K in the pollen tube cytoplasm and associated with pollen tube tonoplast membranes (Lind et al., 1996; Goldraij et al., 2006). Goldraij et al. (2006) also found S-RNase in the lumen of pollen tube vacuoles. In many cases, S-RNase was found in vacuoles with 120K apparently embedded in the surrounding membrane. S-RNase is also found in vacuoles of incompatible pollen tubes, but the breakdown of these vacuoles late in SI and the concomitant release of S-RNase may contribute to the rejection mechanism. Other pistil proteins are also taken up by growing pollen tubes; for example, endocytosis of biotinylated stigma/style Cys-rich adhesin has been reported in lily (Lilium longiflorum) pollen tubes (Kim et al., 2006). Although the uptake of pistil proteins such as 120K and S-RNase has not been well characterized, it is likely that endocytosis and retrograde transport of ECM components occurs on a large scale. Thus, it is important to identify pollen proteins that interact with endocytic cargo from the pistil ECM and that could participate in transport through the pollen tube endomembrane system.We recently described a pollen-specific C2 domain-containing protein, NaPCCP, that interacts with the CTD of the potential cargo proteins, NaTTS and 120K. NaPCCP consists of a short N-terminal domain, an 80-residue C2 domain, and a 79-residue C-terminal region. In vitro pull-down assays showed that the C-terminal region of NaPCCP is sufficient for binding the AGP CTDs (Lee et al., 2008b). Originally implicated in binding mammalian protein kinase C to phosphatidylserine in a calcium-dependent manner (Bazzi and Nelsestuen, 1987, 1990; Brose et al., 1992), C2 domains are now known to contribute to transient membrane association of a variety of proteins with functions that include vesicular transport, lipid modification, GTPase regulation, ubiquitylation, and protein phosphorylation (Coussens et al., 1986; Clark et al., 1991; Brose et al., 1992; Cullen et al., 1995; Dunn et al., 2004). Calcium-independent lipid binding of C2 domain-containing proteins has also been reported (Damer and Creutz, 1994; Fukuda et al., 1994).Here, we report the lipid-binding properties of NaPCCP and its association with the pollen tube endomembrane system. Lipid overlay and liposome-binding experiments show that NaPCCP specifically binds to phosphatidylinositol 3-phosphate (PI3P). Immunolocalization and live imaging studies of compatible pollen tubes show that NaPCCP is associated with the pollen tube plasma membrane (PM) and with punctate structures in the cytoplasm. In SI, incompatible pollen tubes show altered NaPCCP distributions. We speculate that NaPCCP is involved in the uptake and transport of proteins from the ECM.     

Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]_cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]_cyt of the pollen tubes in vivo, we show that [pH]_cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]_cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]_cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]_cyt and its effect on several components in the context of SI-induced events and PCD.Programmed cell death (PCD) in plants is relatively well documented and characterized (Jones and Dangl, 1996; van Doorn, 2011; van Doorn et al., 2011). There is considerable biochemical evidence for the involvement of caspase-like activities in plant PCD (van Doorn and Woltering, 2005). For example, the vacuolar processing enzyme has YVADase (caspase-1-like) activity (Hatsugai et al., 2004; Rojo et al., 2004; Hara-Nishimura et al., 2005), DEVDase (caspase-3-like) and YVADases are associated with PCD in several plant systems (del Pozo and Lam, 1998; Korthout et al., 2000; Danon et al., 2004), and VEIDase (caspase-6-like) is the main caspase-like activity involved in embryonic pattern formation (Bozhkov et al., 2004). However, because plants have no caspase gene homologs (Sanmartín et al., 2005), the nature of their caspase-like enzymes is the subject of considerable debate. Vacuolar cell death is one of two major classes of PCD in plants (van Doorn et al., 2011). It is thought that collapse of the vacuole is a key irreversible step in several plant PCD systems, including during tissue and organ formation, such as the classic differentiation of tracheary elements (Hara-Nishimura and Hatsugai, 2011). Exactly how this is achieved and what processes are involved remain unknown. Until very recently, it was generally thought that the rupturing vacuole releases proteases, hydrolases, and nucleases, allowing cellular disassembly by an autophagy-like process. Some PCD systems cannot be assigned to either class; these include PCD triggered by the hypersensitive response to biotrophic pathogens, PCD in cereal endosperm, and self-incompatibility (SI)-induced PCD (van Doorn et al., 2011).SI is a genetically controlled pollen-pistil cell-cell recognition system. Self-pollen is recognized by the stigma as being genetically identical, resulting in inhibition of pollen tube growth. Most SI systems use tightly linked polymorphic genes: the pollen (male) and pistil (female) S-determinants. In field poppy (Papaver rhoeas), the S-determinants are a 14-kD signaling ligand field poppy stigma S (PrsS) and a unique transmembrane protein field poppy pollen S (PrpS; Foote et al., 1994; Wheeler et al., 2010). These interact in an S-specific manner, and increases in cytosolic free calcium ([Ca2+]_cyt) are triggered in incompatible pollen tubes (Franklin-Tong et al., 1993), resulting in phosphorylation of soluble inorganic pyrophosphatases (sPPases; Rudd et al., 1996; de Graaf et al., 2006), activation of a Mitogen-Activated Protein Kinase (MAPK; Rudd et al., 2003), and increases in reactive oxygen species (ROS) and nitric oxide (Wilkins et al., 2011, 2014). Most of these components are integrated into a signaling network leading to PCD (Bosch et al., 2008; Wilkins et al., 2014). The actin cytoskeleton is a key target in the field poppy SI response, undergoing depolymerization (Snowman et al., 2002) followed by polymerization into highly stable F-actin foci decorated with the actin binding proteins (ABPs) Actin-Depolymerizing Factor (ADF) and Cyclase-Associated Protein (CAP; Poulter et al., 2010, 2011), with both processes being involved in mediating PCD (Thomas et al., 2006). A major player in SI-mediated PCD is a caspase-3-like/DEVDase-like activity (Thomas and Franklin-Tong, 2004; Bosch and Franklin-Tong, 2007). The SI-induced caspase-3-like/DEVDase exhibits maximum substrate cleavage in vitro at pH 5, with peak activity 5 h after SI induction in vivo (Bosch and Franklin-Tong, 2007). The low pH optimum for this caspase-3-like/DEVDase activity is unusual, because most of the cytosolic plant caspase-like activities identified to date have optimal activity close to normal physiological pH (approximate pH, 6.5–7.0; Korthout et al., 2000; Bozhkov et al., 2004; Coffeen and Wolpert, 2004). Because the SI-induced cytosolic-located DEVDase requires a low pH for activity, this suggested that, during SI, the pollen tube cytosol undergoes dramatic acidification. In vivo pH measurements of the cytosol at 1 to 4 h after SI induction confirmed this, when cytosolic pH ([pH]_cyt) had dropped from pH 6.9 to pH 5.5 (Bosch and Franklin-Tong, 2007). This fits the in vitro pH optimum of the caspase-3-like/DEVDase almost exactly, implicating pollen cytosolic acidification as playing a vital role in creating optimal conditions for the activation of the caspase-3-like/DEVDase-like activity and progression of PCD.Under normal cellular conditions, [pH]_cyt is between approximately 6.9 and 7.5 (Kurkdjian and Guern, 1989; Felle, 2001). Pollen tubes, like other tip-growing cells, have [pH]_cyt gradients (Gibbon and Kropf, 1994; Feijó et al., 1999). The [pH]_cyt of the pollen tube shank is an approximate pH of 6.9 to 7.11 (Fricker et al., 1997; Messerli and Robinson, 1998). There has been much debate about the [pH]_cyt gradient, comprising an apical domain with an approximate pH of 6.8 and a subapical alkaline band with an approximate pH of 7.2 to 7.8 in Lilium longiflorum and Lilium formosanum pollen tubes (Fricker et al., 1997; Messerli and Robinson, 1998; Feijó et al., 2001; Lovy-Wheeler et al., 2006). Oscillations of [pH]_cyt between approximate pH values of 6.9 and 7.3 have been linked to tip growth in L. formosanum pollen tubes (Lovy-Wheeler et al., 2006). The vacuole and the apoplast have a highly acidic pH between pH 5 and pH 6 (Katsuhara et al., 1989; Feijó et al., 1999). The majority of studies of pH changes in plant cells reports modest, transient changes in [pH]_cyt of approximately 0.4 and 0.7 pH units during development, gravitropic responses, decreases in light intensity, and addition of elicitors, hormones, and other treatments. For example, during root hair development in Arabidopsis (Arabidopsis thaliana), root [pH]_cyt was elevated from an approximate pH of 7.3 to 7.7 (Bibikova et al., 1998). Root gravitropic responses stimulate small transient [pH]_cyt alterations (Scott and Allen, 1999; Fasano et al., 2001; Johannes et al., 2001). More recently, it has been shown that the [pH]_cyt drops during PCD controlling root cap development; however, exactly how many units the [pH]_cyt decreased was not measured (Fendrych et al., 2014). Other studies investigating [pH]_cyt in response to physiologically relevant signals also report small transient alterations. Light-adapted cells respond to a decrease in light intensity with a rapid transient cytosolic acidification by approximately 0.3 pH units (Felle et al., 1986). Addition of nodulation factors resulted in an increase of 0.2 pH units in root hairs (Felle et al., 1998), and abscisic acid increased the [pH]_cyt of guard cells by 0.3 pH units (Blatt and Armstrong, 1993). Changes in [pH]_cyt are thought to activate stress responses (Felle, 2001). Elicitor treatments resulted in a [pH]_cyt drop of between 0.4 and 0.7 pH units in suspension cells (Mathieu et al., 1996; Kuchitsu et al., 1997), a drop of 0.2 pH units in Nitellopsis obtusa cells treated with salt (Katsuhara et al., 1989), and a drop of 0.3 to 0.7 pH units in Eschscholzia californica (Roos et al., 1998).Here, we investigate SI-induced acidification of the cytosol, providing measurements of physiologically relevant temporal alterations in [pH]_cyt, and identify key targets of this, providing mechanistic insights into these events. The SI-induced acidification plays a pivotal role in the activation of a caspase-3-like/DEVDase activity, the formation of punctate F-actin foci, and ABP localization during SI PCD. We investigate the vacuole as a potential contributor to SI-induced [pH]_cyt acidification.     

Elevation of Pollen Mitochondrial DNA Copy Number by WHIRLY2: Altered Respiration and Pollen Tube Growth in Arabidopsis

In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information.Reflecting their endosymbiotic origin, mitochondria contain DNA genomes (mtDNA) encoding several key proteins for oxidative phosphorylation. As most genes identified in the mitochondrial genome are indispensable for mitochondrial function, it is generally believed that each mitochondrion must possess at least one full copy of the genome. Indeed, this seems to be the case in animals. For example, although the number of mitochondria per cell varies in human, mouse, rabbit, and rat cell lines, the mtDNA copy number per mitochondrion remains constant at 2.6 ± 0.3 (Robin and Wong, 1988). Also, in mouse egg cells, each mitochondrion contains an estimated one to two copies of the mtDNA (Pikó and Matsumoto, 1976).Plant cells, however, have very few copies of the mtDNA compared with the number of mitochondria. For example, in the Cucurbitaceae, cells containing 110 to 140 copies of the mtDNA have 360 to 1,100 mitochondria (Bendich and Gauriloff, 1984). In Arabidopsis (Arabidopsis thaliana), leaf cells each contain approximately 670 mitochondria (Sheahan et al., 2005) and approximately 50 copies of the mtDNA (Draper and Hays, 2000). Thus, in plant cells, each mitochondrion does not possess one complete copy of the mtDNA, a phenomenon that occurs commonly in somatic cells of plants (Preuten et al., 2010). In addition, work in Arabidopsis, barley (Hordeum vulgare), and tobacco (Nicotiana tabacum) showed that cells in leaves, stems, and roots contain few copies of the mtDNA (40–160), whereas cells in root tips contain more copies (300–450; Preuten et al., 2010). This is consistent with the mitochondrial nucleoid diminishment previously observed in developing root and shoot tips (Fujie et al., 1993, 1994), which suggests that the low copy numbers in plant cells result from a decrease in the mtDNA copy number in nondividing cells during development.One question raised by these findings is whether some mitochondria have complete mtDNAs while others have no mtDNA or whether mitochondria have partial mtDNAs. Using techniques for the direct visualization of small amounts of DNA, our group revealed that up to two-thirds of mitochondria in Arabidopsis mesophyll cells totally lack mtDNA and the remaining one-third of mitochondria possess mtDNA of about 100 kb on average (Wang et al., 2010). This agrees well with a previously reported value for mtDNA copy number (about 50 copies per cell; Draper and Hays, 2000) and is consistent with the idea that plant mitochondrial genomes exist as submolecules smaller than the total genomic sizes (Satoh et al., 1993; Kubo and Newton, 2008). Among plant cells possessing low mtDNA copy numbers, the vegetative cell in the pollen grains is an extreme case; a mature pollen grain of Antirrhinum majus, containing many more mitochondria than a somatic cell, possesses only 16 copies of the mtDNA (Wang et al., 2010). Similar to the changes observed in somatic cells, this extremely low level of mtDNA in pollen vegetative cells results from a rapid decrease in mtDNA copy number during pollen development (Sodmergen et al., 1991; Nagata et al., 1999). In A. majus, the vegetative cell in its initial developmental stage has 482.7 copies of the mtDNA per cell, indicating a 30-fold decrease (482.7/16) during development (Wang et al., 2010). These results from both somatic and reproductive cells led to the intriguing idea that the mtDNA copy number in plants decreases in parallel with cell differentiation, to a very low value, and thus that several mitochondria must share the genetic information carried on a single copy of the mtDNA. Plant cell mitochondria undergo frequent and coupled fusions and divisions, which may explain how mitochondria share this information (Arimura et al., 2004). However, the biological significance of why plant cells lose their mtDNA, and how this benefits these cells, remains unknown. Given that pollen germination, pollen tube elongation, and sperm cell delivery all require energy conversion, the extremely low mtDNA copy numbers, such as in pollen vegetative cells, must not compromise mitochondrial function.The mtDNA copy numbers remain constant in various tissues, however, indicating that cellular mechanisms accurately regulate the levels of mtDNA in relation to cell type (Robin and Wong, 1988; Preuten et al., 2010). In yeast and animals, this regulation involves the core enzymes of mtDNA replication, such as DNA polymerase-γ (Sharief et al., 1999), RNA polymerase (Wanrooij et al., 2008), and mitochondrial helicase (Liu et al., 2009), as well as a group of DNA-binding proteins such as ARS-binding factor2 protein in yeast (Saccharomyces cerevisiae; Newman et al., 1996), MITOCHONDRIAL TRANSCRIPTION FACTOR A (TFAM) in human (Alam et al., 2003), and mitochondrial single-stranded DNA binding protein in Drosophila spp. (Maier et al., 2001). Overexpression of TFAM causes an increase in the mtDNA copy number, and RNA interference of TFAM decreases the mtDNA copy number (Ekstrand et al., 2004; Kanki et al., 2004). Also, the homozygous knockout of TFAM in mouse results in embryos that lack mtDNA and thus fail to survive (Larsson et al., 1998). Clearly, protein factors within mitochondrial nucleoids play a crucial role in the regulation of mtDNA copy number.Recent investigation in Arabidopsis revealed that, similar to the case in yeast and animal cells, DNA polymerase, the core enzyme of mtDNA replication, functions to maintain mtDNA levels in plants. Mutation of Arabidopsis PolIA or PolIB (homologs of bacterial DNA polymerase I) causes a reduction in mtDNA copy number, and double mutation of these proteins is lethal (Parent et al., 2011). Also, an Mg²⁺-dependent exonuclease, DEFECTIVE IN POLLEN ORGANELLE DNA DEGRADATION1 (DPD1), degrades organelle DNA, helping to produce the proper amounts of mtDNA in pollen cells (Matsushima et al., 2011; Tang et al., 2012). These results provide insights into the molecular control of mtDNA levels in plants, via both mtDNA replication and mtDNA degradation. Except for these enzymes, however, other protein factors (such as TFAM in animals) have not been identified in plants. The DNA-binding proteins, such as MutS Homolog1 (MSH1), Organellar Single-Strand DNA Binding Protein1 (OSB1), Recombinase A1 (RecA1), RecA3, and WHIRLY2 (WHY2), identified so far in plant mitochondria likely participate in genomic maintenance by affecting substoichiometric shifting (Abdelnoor et al., 2003), stoichiometric transmission (Zaegel et al., 2006), genomic stability (Shedge et al., 2007; Odahara et al., 2009), and DNA repair (Cappadocia et al., 2010). None of these plant nucleoid factors (DNA-binding proteins) has been implicated in the control of mtDNA copy number; thus, the mechanisms by which nonenzyme protein factors regulate mtDNA copy number in plants remain obscure.To test whether nucleoid DNA-binding proteins can affect mtDNA copy number, we examined the effect of producing Arabidopsis WHY2, a single-stranded DNA-binding protein (Cappadocia et al., 2010), in the pollen vegetative cell, which generally does not express WHY2 (Honys and Twell, 2004). We found that expression of WHY2 resulted in a 10-fold increase in mtDNA copy number in the pollen vegetative cell. This increase affected mitochondrial respiration, mitochondrial size, and pollen tube growth. Thus, our results uncover a novel function for WHY2, a member of the plant Whirly protein family, in regulating mtDNA amounts and indicate that, in plants, low mtDNA copy number does not compromise mitochondrial function but rather promotes proper mitochondrial function.     

NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes

In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.To avoid low-fitness progeny, many plants have developed a cell-cell interaction mechanism to promote outcrossing, through the recognition and discrimination of both self and nonself pollen. This recognition system is controlled by the highly polymorphic self-incompatibility S-locus, which determines pollination specificity in both the pollen and pistil. Pollen is rejected when male and female S-haplotypes coincide (de Nettancourt, 1977, 2001; Franklin et al., 1995).In Solanaceae, Plantaginaceae, and Rosaceae, the S-locus product in the pistil is an extracellular glycoprotein named S-RNase (Anderson et al., 1986; McClure et al., 1989). During pollination, S-RNase is taken up by both compatible and incompatible pollen tubes (Luu et al., 2000) and targeted to a vacuole (Goldraij et al., 2006). In the later stages of an incompatible cross, the S-RNase-containing vacuole is disrupted and the S-RNases are released to the pollen tube cytoplasm, where RNA degradation can occur (McClure et al., 2011).The S-pollen gene encodes an SLF or SFB (SLF/SFB; for S-locus F-box) protein, which is a member of the F-box protein family (Entani et al., 2003; Sijacic et al., 2004). In vitro binding assays show that PiSLF in Petunia inflata physically interacts with S-RNases, although this interaction is stronger with nonself S-RNases than with self S-RNases (Hua and Kao, 2006). Additional protein-protein interaction assays suggest that SLF/SFB may be a component of an SCF (for Skp1-Cullin1-F-box) or SCF-like complex (Qiao et al., 2004; Hua and Kao, 2006). Notably, data from Zhao et al. (2010) in Petunia hybrida show that reduction of PhSSK1 (for P. hybrida SLF-interacting Skp-like1) and its Antirrhinum hispanicum ortholog, AhSSK1, is also required for cross-pollen compatibility.Although S-RNase and SLF/SFB define pollen rejection S-specificity, modifier genes unlinked to the S-locus are required for self-incompatibility (SI; Martin, 1968; Ai et al., 1991; Murfett et al., 1996; Tsukamoto et al., 1999).To date, only two pistil-modifier genes have been identified: High Top-Band (HT-B) and 120K. In Nicotiana spp., HT-B is an 8.6-kD acidic protein with a domain consisting of 20 Asn and Asp residues toward its C terminus (McClure et al., 1999; Kondo and McClure, 2008). Loss-of-function assays prove HT-B to be essential for pollen rejection in Nicotiana spp., Solanum spp., and Petunia spp. (McClure et al., 1999; Kondo et al., 2002; O’Brien et al., 2002; Sassa and Hirano, 2006; Puerta et al., 2009), although it is not expressed in SI Solanum habrochaites, prompting the speculation that in this species a related gene, HT-A, may function as a substitute (Covey et al., 2010). Immunolocalization shows that HT-B is readily taken up by pollen tubes during pollination. Its steady-state levels decrease slightly in pollen tubes from incompatible pollinations. However, in compatible crosses, HT-B levels decrease 75% to 97%, probably as a result of protein degradation (Goldraij et al., 2006).120K is a style-specific 120-kD arabinogalactan protein (Schultz et al., 1997) that is taken up by pollen tubes (Lind et al., 1996) and appears to be associated with S-RNase-containing vacuoles (Goldraij et al., 2006). 120K forms complexes with S-RNases and other proteins (Cruz-Garcia et al., 2005) in vitro, and suppression of 120K expression prevents S-specific pollen rejection (Hancock et al., 2005). Protein-protein interaction assays demonstrate that 120K interacts with the pollen-specific protein NaPCCP (a pollen C2 domain-containing protein), a protein that binds phosphatidylinositol 3-phosphate and is associated with the pollen tube endomembrane system (Lee et al., 2008, 2009).Two models have been proposed to explain pollen rejection in Solanaceae. (1) The S-RNase degradation model (Hua and Kao, 2006; Hua et al., 2007, 2008; Kubo et al., 2010) focuses on S-RNase-SLF interactions that bring about preferential nonself S-RNase degradation. In this model, strong nonself S-RNase-SLF interactions lead to the degradation of nonself S-RNases by the ubiquitin-26S proteasome system, allowing pollen tubes to escape from its cytotoxic effect. Weak self S-RNase-SLF interactions, in contrast, permit the persistence of sufficient free S-RNase that pollen tube RNA is degraded, resulting in self-pollen rejection. Notably, by functional and protein-protein interaction assays in Petunia spp., Kubo et al. (2010) found at least three types of divergent SLF proteins encoded at the S-locus, each recognizing a subgroup of nonself S-RNases. The authors proposed the collaborative nonself recognition model, where multiple SLF proteins interact with nonself S-RNases to protect nonself pollen from degradation (Kubo et al., 2010). (2) The compartmentalization model incorporates the observations that pollen tubes internalize both self and nonself S-RNases and targets them to vacuoles and that HT-B is degraded in compatible crosses but is stable in incompatible crosses (Goldraij et al., 2006). In incompatible crosses, the S-RNase-containing vacuoles are ultimately disrupted and S-RNases are released to the cytoplasm, where they degrade RNA, leading to rejection of self-pollen. In compatible crosses, the integrity of the S-RNase-containing vacuoles is preserved, allowing pollen tube growth to continue. Thus, in this model, self or nonself S-RNase-SLF interactions determine the specificity of pollen rejection indirectly.Biochemical and genetic data show that pistil-modifier genes apart from HT-B and 120K are required for SI. We recently described NaStEP (for N. alata Stigma-Expressed Protein), an abundant, pistil-specific stigma protein found in SI Nicotiana spp. (Busot et al., 2008). Its abundance in SI species made NaStEP a strong modifier gene candidate. Here, we demonstrate that NaStEP is taken up by pollen tubes, has subtilisin inhibitory activity, and that suppressing its expression in transgenic hybrids disrupts pollen rejection. Moreover, when NaStEP-suppressed hybrids are pollinated, HT-B protein is degraded in both compatible and incompatible pollen tubes, while in wild-type SI N. alata, HT-B is preferentially stabilized in incompatible pollen tubes.     

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis

Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance.In flowering plants, sexual reproduction occurs as a result of constant communication between the male gametophyte and the female reproductive organ, from the initial acceptance of compatible pollen to final step of successful fertilization (for review, see Beale and Johnson, 2013; Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015). In the Brassicaceae, the stigmas that present a receptive surface for pollen are categorized as dry and covered with unicellular papillae (Heslop-Harrison and Shivanna, 1977). Communication is initiated rapidly following contact of a pollen grain with a stigmatic papilla, as the role of the papillae is to regulate the early cellular responses leading to compatible pollen germination. The basal compatible pollen recognition response also presents a barrier to foreign pollen or is inhibited with self-incompatible pollen (for review, see Dickinson, 1995; Hiscock and Allen, 2008; Chapman and Goring, 2010; Indriolo et al., 2014b).The initial adhesive interaction between the pollen grain and the papilla cell in the Brassicaceae is mediated by the exine of the pollen grain and the surface of the stigmatic papilla (Preuss et al., 1993; Zinkl et al., 1999). A stronger connection results between the adhered pollen grain and the stigmatic papilla with the formation of a lipid-protein interface (foot) derived from the pollen coat and the stigmatic papillar surface (Mattson et al., 1974; Stead et al., 1980; Gaude and Dumas, 1986; Elleman and Dickinson, 1990; Elleman et al., 1992; Preuss et al., 1993; Mayfield et al., 2001). It is at this point that a Brassicaceae-specific recognition of compatible pollen is proposed to occur (Hülskamp et al., 1995; Pruitt, 1999), though the nature of this recognition system is not clearly defined. Two stigma-specific Brassica oleracea glycoproteins, the S-Locus Glycoprotein and S-Locus Related1 (SLR1) protein, play a role in compatible pollen adhesion (Luu et al., 1997, 1999), potentially through interactions with the pollen coat proteins, PCP-A1 and SLR1-BP, respectively (Doughty et al., 1998; Takayama et al., 2000). The simultaneous recognition of self-incompatible pollen would also take place at this stage (for review, see Dresselhaus and Franklin-Tong, 2013; Indriolo et al., 2014b; Sawada et al., 2014). Thus, this interface not only provides a strengthened bond between the pollen grain and stigmatic papilla, but likely facilitates the interaction of signaling proteins from both partners to promote specific cellular responses in the stigmatic papilla toward the pollen grain.One response regulated by these interactions is the release of water from the stigmatic papilla to the adhered compatible pollen grain to enable the pollen grain to rehydrate, germinate, and produce a pollen tube (Zuberi and Dickinson, 1985; Preuss et al., 1993). Upon hydration, the pollen tube emerges at the site of pollen-papilla contact and penetrates the stigma surface between the plasma membrane and the overlaying cell wall (Elleman et al., 1992; Kandasamy et al., 1994). Pollen tube entry into the stigmatic surface represents a second barrier, selecting compatible pollen tubes. Subsequently, the compatible pollen tubes traverse down to the base of the stigma, enter the transmitting tract, and grow intracellularly toward ovules for fertilization. Pollen-pistil interactions at these later stages are also highly regulated (for review, see Beale and Johnson, 2013; Dresselhaus and Franklin-Tong, 2013; Higashiyama and Takeuchi, 2015).EXO70A1, a subunit of the exocyst, was identified as a factor involved in early pollen-stigma interactions, where it is required in the stigma for the acceptance of compatible pollen and inhibited by the self-incompatibility response (Samuel et al., 2009). Stigmas from the Arabidopsis (Arabidopsis thaliana) exo70A1 mutant display constitutive rejection of wild-type-compatible pollen (Samuel et al., 2009; Safavian et al., 2014). This stigmatic defect was rescued by the stigma-specific expression of an Red Fluorescent Protein (RFP):EXO70A1 transgene (Samuel et al., 2009) or partially rescued by providing a high relative humidity environment (Safavian et al., 2014). In addition, the stigma-specific expression of an EXO70A1 RNA interference construct in Brassica napus ‘Westar’ resulted in impaired compatible pollen acceptance and a corresponding reduction in seed production compared with compatible pollinations with wild-type B. napus ‘Westar’ pistils (Samuel et al., 2009). From these studies, EXO70A1 was found to be a critical component in stigmatic papillae to promote compatible pollen hydration and pollen tube entry through the stigma surface. One of the functions of the exocyst is to mediate polar secretion (for review, see Heider and Munson, 2012; Zárský et al., 2013; Synek et al., 2014). Consistent with this, previous studies have observed vesicle-like structures in proximity to the stigmatic papillar plasma membrane in response to compatible pollen in both Brassica spp. and Arabidopsis species (Elleman and Dickinson, 1990, 1996; Dickinson, 1995; Safavian and Goring, 2013; Indriolo et al., 2014a). The secretory activity is predicted to promote pollen hydration and pollen tube entry. As well, consistent with the proposed inhibition of EXO70A1 by the self-incompatibility pathway (Samuel et al., 2009), a complete absence or a significant reduction of vesicle-like structures at the stigmatic papillar plasma membrane was observed in the exo70A1 mutant and with self-incompatible pollen (Safavian and Goring, 2013; Indriolo et al., 2014a).The exocyst is a well-defined complex in yeast (Saccharomyces cerevisiae) and animal systems, consisting of eight subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84 (TerBush et al., 1996; Guo et al., 1999). Exocyst subunit mutants were first identified in yeast as secretory mutants displaying a cytosolic accumulation of secretory vesicles (Novick et al., 1980). Subsequent work defined roles for the exocyst in vesicle docking at target membranes in processes such as regulated secretion, polarized exocytosis, and cytokinesis to facilitate membrane fusion by Soluble NSF Attachment protein Receptor (SNARE) complexes (for review, see Heider and Munson, 2012; Liu and Guo, 2012). In plants, genes encoding all eight exocyst subunits have been identified, and many of these genes exist as multiple copies. For example, the Arabidopsis genome contains single copy genes for SEC6 and SEC8, two copies each for SECRETORY3 (SEC3), SEC5, SEC10, and SEC15, three EXO84 genes, and 23 EXO70 genes (Chong et al., 2010; Cvrčková et al., 2012; Vukašinović et al., 2014). Ultrastructural studies using electron tomography uncovered the existence of a structure resembling the exocyst in Arabidopsis (Otegui and Staehelin, 2004; Seguí-Simarro et al., 2004). Localization studies of specific Arabidopsis exocyst subunits also supported conserved roles in polarized exocytosis and cytokinesis in plants. Localization studies have shown EXO70, SEC6, and SEC8 at the growing tip of pollen tubes (Hála et al., 2008), EXO70A1 at the stigmatic papillar plasma membrane (Samuel et al., 2009), SEC3a, SEC6, SEC8, SEC15b, EXO70A1, and EXO84b at the root epidermal cell plasma membrane and developing cell plate (Fendrych et al., 2010, 2013; Wu et al., 2013; Zhang et al., 2013; Rybak et al., 2014), and SEC3a at the plasma membrane in the embryo and root hair (Zhang et al., 2013). Similar to the yeast exocyst mutants, vesicle accumulation has also been observed in the exo70A1 and exo84b mutants (Fendrych et al., 2010; Safavian and Goring, 2013). Taken together, these findings strongly support that plant exocyst subunits function in vivo in vesicle docking at sites of polarized secretion and cytokinesis (for review, see Zárský et al., 2013). In support of this, a recent study investigating Transport Protein Particle (TRAPP)II and exocyst complexes during cytokinesis in Arabidopsis has identified all eight exocyst components in immunoprecipitated complexes (SEC3a/SEC3b, SEC5a, SEC6, SEC8, SEC10, SEC15b, EXO70A1, EXO70H2, and EXO84b; Rybak et al., 2014).Several plant exocyst subunit genes have been implicated in biological processes that rely on regulated vesicle trafficking, where corresponding mutants have displayed a range of growth defects. At the cellular level, these phenotypes have been associated with decreased cell elongation and polar growth (Cole et al., 2005, 2014; Wen et al., 2005; Synek et al., 2006), defects in cytokinesis and cell plate formation (Fendrych et al., 2010; Wu et al., 2013; Rybak et al., 2014), and disrupted Pin-Formed (PIN) auxin efflux carrier recycling and polar auxin transport (Drdová et al., 2013). Several Arabidopsis subunit mutants display strong growth defects such as the sec3a mutant with an embryo-lethal phenotype (Zhang et al., 2013), sec6, sec8, and exo84b mutants with severely dwarfed phenotypes and defects in root growth (Fendrych et al., 2010; Wu et al., 2013; Cole et al., 2014), and exo70A1 with a milder dwarf phenotype (Synek et al., 2006). The Arabidopsis exo70A1 mutant has also been reported to have defects in root hair elongation, hypocotyl elongation, compatible pollen acceptance, seed coat deposition, and tracheary element differentiation (Synek et al., 2006; Samuel et al., 2009; Kulich et al., 2010; Li et al., 2013). Essential roles for other exocyst subunits include Arabidopsis SEC5a/SEC5b, SEC6, SEC8, and SEC15a/SEC15b in male gametophyte development and pollen tube growth (Cole et al., 2005; Hála et al., 2008; Wu et al., 2013), SEC8 in seed coat deposition (Kulich et al., 2010), SEC5a, SEC8, EXO70A1, and EXO84b in root meristem size and root cell elongation (Cole et al., 2014), and a maize (Zea mays) SEC3 homolog in root hair elongation (Wen et al., 2005). Finally, the Arabidopsis EXO70B1, EXO70B2, and EXO70H1 subunits have been implicated in plant defense responses (Pecenková et al., 2011; Stegmann et al., 2012; Kulich et al., 2013; Stegmann et al., 2013).Even with these detailed studies on the functions of exocyst subunits in plants, a systematic demonstration of the requirement of all eight exocyst subunits in a specific plant biological process is currently lacking. EXO70A1 was previously identified as an essential factor in the stigma for compatible pollen-pistil interactions in Arabidopsis and B. napus (Samuel et al., 2009), and we hypothesized that this protein functions as part of the exocyst complex to tether post-Golgi secretory vesicles to stigmatic papillar plasma membrane (Safavian and Goring, 2013). To provide support for the proposed biological role of the exocyst in the stigma for compatible pollen acceptance, we investigated the roles of the remaining seven subunits, SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmatic papillae. Given that some Arabidopsis exocyst subunits were previously determined to be essential at earlier growth stages, stigma-specific RNA-silencing constructs were used for each exocyst subunit, and the early postpollination stages were analyzed for these transgenic lines. Our collective data demonstrates that all eight exocyst subunits are required in the stigma for the early stages of compatible pollen-pistil interactions.     

ECHIDNA Protein Impacts on Male Fertility in Arabidopsis by Mediating trans-Golgi Network Secretory Trafficking during Anther and Pollen Development

The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-YELLOW FLUORESCENT PROTEIN) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.Pollen production and release is a critical stage in plant development that typically involves gene expression from over half of the genome. The extent of genomic involvement in pollen development is illustrated by the high frequency of mutations that result in a failure of male fertility; these can be a consequence of the failure of pollen development or pollen release, dehiscence. Detailed analysis of male-sterile mutants in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has improved the basic understanding of pollen and anther development (Scott et al., 2004; Ma, 2005; Wilson and Zhang, 2009; Cui et al., 2012); however, there are multiple aspects of pollen formation that are still unclear, and many defects result in uncharacterized effects of reduced fertility or complete sterility.The ECHIDNA (ECH) gene was initially identified from expression profiling of the vascular cambium in poplar (Populus spp.) and associated with secondary xylem formation (Hertzberg et al., 2001). The Arabidopsis ech mutant was shown to have a bushy stature with defects in root and hypocotyl elongation, which was linked to defective cell expansion and elongation (Gendre et al., 2011). Analysis of roots in the ech mutant and complementation analyses in yeast showed that the ECH protein impacts on cell expansion by mediating trans-Golgi network (TGN) secretory trafficking but does not affect endocytosis (Gendre et al., 2011). However, in addition to the defects associated with plant stature, the ech mutant also displays a previously unreported phenotype of reduced fertility.Pollen development occurs in a specialized organ, the stamen, which comprises anthers that hold the developing pollen supported by a filament containing the vasculature connections. Stamen primordia arise from divisions in the L1, L2, and L3 layers in the floral meristem. Divisions in the L2 layer result in four clusters of archesporial cells that subsequently form the central sporogenous cells, which are surrounded by four maternal cell layers: the tapetum, middle cell layer, endothecium, and outer epidermis (Scott et al., 2004). The structure of the maternal anther cell layers has been shown to be critical for the production and release of functional pollen, as demonstrated in a number of male-sterile mutants, which have defects in cell division and early stages of differentiation of the tapetum and sporogenous cells. For example, mutants of the Leu-rich repeat receptor kinase EXTRA SPOROGENOUS CELLS (EXS)/EXCESS MICROSPOROCYTES1 (Canales et al., 2002; Zhao et al., 2002) and its ligand TAPETAL DETERMINANT1 (Jia et al., 2008) result in sterility due to the formation of additional male sporocytes and a lack of tapetal cells.The tapetum has been shown to be critical for functional pollen formation, with many of the characterized male-sterile mutants exhibiting abnormal tapetal development, including DYSFUNCTIONAL TAPETUM1 (DYT1; Zhang et al., 2006; Zhu et al., 2008), TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1; Zhu et al., 2008), ABORTED MICROSPORES (AMS; Sorensen et al., 2003; Xu et al., 2010), and MALE STERILITY1 (MS1; Wilson et al., 2001; Ito and Shinozaki, 2002). After differentiation, the tapetum layer becomes metabolically highly active and plays an essential role in the biosynthesis and secretion of sporopollenin and other wall materials for the developing pollen, prior to breakdown via programmed cell death (Ariizumi and Toriyama, 2011). A frequently observed phenotype in male-sterile mutants is enlarged tapetal cells that show defects in secretion and subsequent alterations in programmed cell death breakdown (Wilson and Zhang, 2009). This indicates the important role that the tapetum plays in the regulation of pollen development and, in particular, the passage of materials to the central locule for viable pollen production.Male-sterile phenotypes have also been identified due to a failure of pollen release, dehiscence. Secondary thickening occurs specifically in the endothecium layer of the anther; this layer and the presence of selective thickening within it are critical to generate the differential forces that are required for anther dehiscence and pollen release (Wilson et al., 2011; Nelson et al., 2012). The importance of this secondary thickening is demonstrated in the myb26 mutant (Yang et al., 2007) and in the double NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (nst1 nst2) mutant (Mitsuda et al., 2005), which lack endothecium thickening and, as a result, fail to dehisce (Nelson et al., 2012).Previous investigations of the ech mutation indicated that it is impaired in TGN secretion, resulting in dwarf plants with defects in root and hypocotyl cell elongation. The ech mutant also has an uncharacterized phenotype of impaired male fertility; therefore, a detailed analysis of reproduction in the ech mutant was conducted. ECH expression was seen in the anther tapetum during the early stages of tapetal development and microspore release but was subsequently detected in the pollen, pollen tube, and stylar tissues. The reduced fertility was linked to decreased anther size and pollen production but also to reductions in pollen viability, anther opening, and pollen tube growth. The anther wall thickening was reduced and disorganized in ech, possibly as a consequence of altered secretion of wall materials through the TGN. The male-sterile myb26 mutant has defects in anther endothecium wall thickening resulting in a failure of dehiscence; the ech myb26 double mutant exhibits the phenotypes of both mutants and fails to produce secondary thickening, indicating that the ECH-mediated pathway is acting independently of or upstream through MYB26, possibly by providing the components required for secondary cell wall thickening. The reduction in male fertility, therefore, is likely to be a consequence of multiple effects due to altered secretion in the anther because of impaired TGN transport in the ech mutant; the resulting defects are associated with tapetum and pollen wall development but also anther dehiscence and pollen tube formation.