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.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP’s activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

The delivery of nonmotile sperm to the embryo sac via a pollen tube is a key innovation that allowed flowering plants to carry out sexual reproduction without the need for water (Friedman, 1993; Lord and Russell, 2002). Both the speed and signal responsiveness of pollen tube growth are critical for successful fertilization (Johnson et al., 2019). The typical shape of a growing pollen tube cell protruding from a pollen grain is a cylinder with a dome-shaped tip (Geitmann, 2010). Maintaining such a typical tube shape during pollen tube growth is fundamental to support its ability for fast growth (Michard et al., 2017), and a plasticity range of tubular growth rates allows a pollen tube to optimize directional growth along its journey from the stigma to the ovule (Luo et al., 2017). The pollen tube cell extends mainly by tip growth, requiring huge amounts of secretion/exocytosis at the tip (McKenna et al., 2009; Grebnev et al., 2017). The newly secreted cell wall at the tip is mainly composed of esterified pectin, which is expandable, whereas cell wall remodeling at the lateral region (including pectin deesterification and callose deposition) limits expansion (Grebnev et al., 2017). The tip width of a growing pollen tube actually reflects the size of the secretion zone capped by an expandable membrane and cell wall, as a collective result of multiple pollen tube growth machineries (Luo et al., 2017).The tip-localized exocytosis of a growing pollen tube is supported by a spatiotemporal tightly controlled actin cytoskeleton network (Hepler, 2016). The actin cytoskeleton configuration in a pollen tube includes highly dynamic fine actin filaments in the apical and subapical regions and parallel longitudinal actin bundles in the shank region (Qu et al., 2017). Various actin-binding proteins, such as actin nucleation factors, actin-severing proteins, and actin-bundling factors, are responsible for organizing the dynamic actin cytoskeleton network (Ren and Xiang, 2007). For example, the actin-bundling proteins fimbrin and LIM (Lin-1, isl1, Mec3) domain-containing proteins function in shank-localized actin bundles in pollen tubes (Zhang et al., 2019). For another example, the actin nucleator formin (formin3 in Arabidopsis [Arabidopsis thaliana] and formin1 in lily [Lilium longiflorum]) functions in actin polymerization in the pollen tube tip (Li et al., 2017; Lan et al., 2018). The branched actin nucleator Actin-Related Protein2/3 (ARP2/3) complex is an evolutionarily conserved, seven-subunit complex consisting of the actin-related proteins ARP2 and ARP3 (Machesky et al., 1994). The ARP2/3 complex initiates the formation of branches on the side of preexisting actin filaments, locally creating a force-generating branched actin network that underlies cellular protrusion and movement (Blanchoin et al., 2000; Amann and Pollard, 2001; Molinie and Gautreau, 2018). The phenotypes of mutants in ARP2/3 in the moss Physcomitrella patens (Harries et al., 2005; Perroud and Quatrano, 2006), in Arabidopsis (Le et al., 2003; Li et al., 2003; Mathur et al., 2003; Brembu et al., 2004; Deeks et al., 2004), in maize (Zea mays; Frank and Smith, 2002), and in tomato (Solanum lycopersicum; Chang et al., 2019) demonstrated the broad importance of the ARP2/3 complex and its activation during cellular morphogenesis, including tip-growing cells. Perhaps surprisingly, in Arabidopsis, null ARP2/3 alleles are transmitted normally through pollen and there is no obvious root hair phenotype (Le et al., 2003; Djakovic et al., 2006).These cell growth machineries are tightly coordinated by multiple signaling pathways, including membrane-localized receptor kinases and Rho-of-Plants (ROP) GTPases (Li et al., 2018). The tomato pollen-specific and membrane-localized receptor kinases LePRK1 and LePRK2 mediate signaling during pollen tube growth (Muschietti et al., 1998). LePRK2 perceives several extracellular growth-stimulating factors, including a Cys-rich extracellular protein (Late-Anther-Specific52 [LAT52]), a Leu-rich repeat protein from pollen, and two pistil/stigma molecules, Style Interactor for LePRKs and Stigma-Specific Protein1 (Tang et al., 2002, 2004; Wengier et al., 2003, 2010), which increase the speed of pollen tube growth (Zhang et al., 2008b; Huang et al., 2014). LePRK2 antisense and RNA interference (RNAi) pollen tubes grow slower (Zhang et al., 2008b), consistent with a positive role for LePRK2 in regulating the speed of pollen tube growth. LePRK1 binds LePRK2 (Wengier et al., 2003), but LePRK1 plays a negative role in pollen tube growth by controlling a switch from a fast tubular mode to a slow blebbing mode (Gui et al., 2014). LePRK1 RNAi pollen tubes burst more often than wild-type pollen tubes, implicating a role for LePRK1 in maintaining plasma membrane integrity (Gui et al., 2014). An Arabidopsis paralog of these LePRKs, PRK6, also localized on the tip membrane, perceives Arabidopsis attraction cues from the female, AtLURE1s, to guide pollen tube growth (Takeuchi and Higashiyama, 2016; Zhang et al., 2017).Rho family small guanine nucleotide-binding proteins called ROPs or RACs, which can switch between a GDP-bound inactive form and a GTP-bound active form, are regulators of polar growth in pollen tubes (Cheung and Wu, 2008; Yang, 2008). In Arabidopsis, ROP1-dependent signaling controls tip growth. Active ROP1 defines a cap region in the apical plasma membrane as an exocytosis zone (Luo et al., 2017). Overexpression of ROP1 or of a constitutively active version resulted in pollen tube tip swelling (i.e. increased tip width) and slower growth (i.e. reduced tube length), while overexpressing a dominant negative version of ROP1 inhibited pollen tube growth (i.e. shorter but normal width tubes). The size of the pollen tube tip reflects the aggregate activity of membrane-associated ROP at the tip (McKenna et al., 2009; Luo et al., 2017). Tomato ROPs have been reported to be associated with the LePRK1-LePRK2 complex (Wengier et al., 2003) and therefore presumably play similar roles as the Arabidopsis homologs in pollen tube growth, yet their biological roles have not been directly investigated.Guanine nucleotide exchange factors (GEFs) activate ROPs by promoting the conversion of ROP/RAC GTPases from a GDP-bound inactive form to a GTP-bound active form. Plants possess a plant-specific ROPGEF family whose members contain a highly conserved GEF catalytic domain, the PRONE (plant-specific ROP nucleotide exchanger) domain (Berken et al., 2005; Gu et al., 2006). The intracellular portions of LePRK1 and LePRK2 interact with Kinase Partner Protein (KPP; Kaothien et al., 2005), whose Arabidopsis homologs were later shown to belong to the PRONE-type ROPGEF family (Berken et al., 2005; Gu et al., 2006). Pollen tubes overexpressing nearly full-length KPP (missing eight amino acids at the N terminus) developed swollen tips with abnormal cytoplasmic streaming and F-actin arrangements (Kaothien et al., 2005). An Arabidopsis homolog of receptor kinase, AtPRK2a (also named AtPRK2), interacts with AtROPGEF12 (Zhang and McCormick, 2007) and with AtROPGEF1 (Chang et al., 2013) to affect ROP activity. Based on the in vitro catalytic activity of full-length and truncated AtROPGEF1, an autoinhibition conferred by the C-terminal variable region was proposed (Gu et al., 2006). AtROPGEF12 was also shown to interact with the guidance receptor kinase PRK6 (Takeuchi and Higashiyama, 2016).Increased expression of full-length KPP increased the speed of pollen tube growth without significantly affecting pollen tube shape. We show biochemically that the PRONE domain of KPP does have ROPGEF activity on several class I ROPs, with highest activity on ROP4. The N-terminal domain of KPP inhibits its own GEF activity, while its C-terminal domain enhances its own GEF activity. The C-terminal domain of KPP is also required for its interactions with LePRK1, LePRK2, and an actin-bundling protein, Pollen-expressed LIM2a (PLIM2a), while the C-terminal domain alone is sufficient to bind LePRK1 but insufficient to bind LePRK2. Furthermore, self-associated KPP colocalized with the actin nucleation proteins ARP2/3 complex during pollen tube growth and enriched the membrane localization of ARP2/3 in the pollen tube. Interfering with ARP2/3 activation by coexpressing a dominant negative version of ARP2 reduced the speed of pollen tube growth and alleviated the defects caused by the overexpression of truncated KPP. CK-666, a specific small molecule inhibitor of ARP2/3 activation, canceled the promotive effect of full-length KPP on the speed of pollen tube growth. These results indicate that during pollen germination and tube growth, KPP not only links pollen receptor kinase and ROP signaling but also links the actin network to the pollen tube plasma membrane, thereby directly affecting the cellular morphology and efficiency of pollen tube growth.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

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.