LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.     

New pollen-specific receptor kinases identified in tomato,maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.     

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.     

Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth

The pollen-specific receptor kinases LePRK1 and LePRK2 have localization and expression profiles that strongly suggest they play roles in pollen germination and tube growth. To identify downstream components of LePRK signaling, we used their cytoplasmic domains (CDs) as baits in yeast two-hybrid screens of a tomato pollen cDNA library. A pollen-specific protein we named kinase partner protein (KPP) interacted with the CDs of both LePRK1 and LePRK2 in yeast and in an in vitro pull-down assay, and with LePRK2 in a co-immunoprecipitation assay. KPP is a peripheral membrane protein and is phosphorylated in pollen. Pollen tubes over-expressing KPP developed balloon-like tips with abnormal cytoplasmic streaming and F-actin arrangements and plants over-expressing KPP exhibited impaired transmission of the transgene through the male. KPP-like genes are found only in plants; the 14 family members in Arabidopsis thaliana exhibit diverse expression patterns and potentially play roles in signaling pathways in other tissues.     

Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth

SHY, a pollen-specific gene identified in a screen for genes upregulated at pollen germination, encodes a leucine-rich repeat (LRR) protein that is predicted to be secreted. To test if SHY plays an important role during pollen germination, we generated transgenic plants expressing an antisense (AS) copy of the SHY cDNA in pollen. Primary transformants exhibited poor seed set, but homozygous lines could be identified. In these lines, nearly all pollen tubes failed to reach the ovules; tube growth was arrested at the apex of the ovary and the pollen tubes exhibited abnormal callose deposits throughout the tube and in the tips. We show that a SHY::eGFP fusion protein is targeted to the cell wall. The structure of the SHY protein is nearly identical to other extracellular matrix glycoproteins that are composed of LRRs, such as the polygalacturonase inhibitor proteins (PGIP) of plants. PGIPs may function as defense proteins by inhibiting fungal endo-polygalacturonases, but enzyme assays with extracts of AS-SHY pollen do not support such an inhibitor role for SHY. The tomato ortholog of SHY interacts with a tomato receptor kinase (LePRK2) in yeast two-hybrid and pull-down assays; this, and the AS-SHY phenotypes, suggest instead that SHY might function in a signal transduction pathway mediating pollen tube growth.     

On your mark,get set,GROW! LePRK2-LAT52 interactions regulate pollen tube growth

Recent discoveries show that LAT52 and LePRK2, two pollen-specific proteins, interact in what might be an autocrine signaling system. This exciting finding indicates that successful fertilization requires ligand-receptor kinase signals that regulate pollen-tube growth. The stage is now set to identify other components of this pathway and to explore their connections with the many signals exchanged between pollen and pistil.     

Overexpression of the Tomato Pollen Receptor Kinase LePRK1 Rewires Pollen Tube Growth to a Blebbing Mode

The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing “blebs.” Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca²⁺-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells.     

Two Arabidopsis AGC kinases are critical for the polarized growth of pollen tubes

Reproduction of flowering plants requires the growth of pollen tubes to deliver immotile sperm for fertilization. Pollen tube growth resembles that of polarized metazoan cells, in that some molecular mechanisms underlying cell polarization and growth are evolutionarily conserved, including the functions of Rho GTPases and the dynamics of the actin cytoskeleton. However, a role for AGC kinases, crucial signaling mediators in polarized metazoan cells, has yet to be shown in pollen tubes. Here we demonstrate that two Arabidopsis AGC kinases are critical for polarized growth of pollen tubes. AGC1.5 and AGC1.7 are pollen-specific genes expressed during late developmental stages. Pollen tubes of single mutants had no detectable phenotypes during in vitro or in vivo germination, whereas those of double mutants were wider and twisted, due to frequent changes of growth trajectory in vitro . Pollen tubes of the double mutant also had reduced growth and were probably compromised in response to guidance cues in vivo . In the agc1.5 background, downregulation of AGC1.7 using an antisense construct phenocopied the growth defect of double mutant pollen tubes, providing additional support for a redundant function of AGC1.5/1.7 in pollen tube growth. Using the actin marker mouse Talin, we show that pollen tubes of double mutants had relatively unaffected longitudinal actin cables but had ectopic filamentous actin, indicating disturbed control of polarity. Our results demonstrate that AGC1.5 and AGC1.7 are critical components of the internal machinery of the pollen tube leading to polarized growth of pollen tubes.     

The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth

The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca²⁺ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca²⁺ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding the control of pollen germination and tube growth. Received: 12 December 1996 / Accepted: 15 January 1997     

Tomato Pistil Factor STIG1 Promotes in Vivo Pollen Tube Growth by Binding to Phosphatidylinositol 3-Phosphate and the Extracellular Domain of the Pollen Receptor Kinase LePRK2

The speed of pollen tube growth is a major determinant of reproductive success in flowering plants. Tomato (Solanum lycopersicum) STIGMA-SPECIFIC PROTEIN1 (STIG1), a small Cys-rich protein from the pistil, was previously identified as a binding partner of the pollen receptor kinase LePRK2 and shown to promote pollen tube growth in vitro. However, the in vivo function of STIG1 and the underlying mechanism of its promotive effect were unknown. Here, we show that a 7-kD processed peptide of STIG1 is abundant in the stigmatic exudate and accumulates at the pollen tube surface, where it can bind LePRK2. Antisense LePRK2 pollen was less responsive than wild-type pollen to exogenous STIG1 in an in vitro pollen germination assay. Silencing of STIG1 reduced both the in vivo pollen tube elongation rate and seed production. Using partial deletion and point mutation analyses, two regions underlying the promotive activity of the STIG1 processed peptide were identified: amino acids 80 to 83, which interact with LePRK2; and amino acids 88 to 115, which bind specifically to phosphatidylinositol 3-phosphate [PI(3)P]. Furthermore, exogenous STIG1 elevated the overall redox potential of pollen tubes in both PI(3)P-dependent and LePRK2-dependent manners. Our results demonstrate that STIG1 conveys growth-promoting signals acting through the pollen receptor kinase LePRK2, a process that relies on the external phosphoinositide PI(3)P.     

STIL,a peculiar molecule from styles,specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth <Emphasis Type="Italic">in vitro</Emphasis>

Background  

LePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.     

Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2.

We screened for pollen-specific kinase genes, which are potential signal transduction components of pollen-pistil interactions, and isolated two structurally related receptor-like kinases (RLKs) from tomato, LePRK1 and LePRK2. These kinases are similar to a pollen-expressed RLK from petunia, but they are expressed later during pollen development than is the petunia RLK. The abundance of LePRK2 increases when pollen germinates, but LePRK1 remains constant. Both LePRK1 and LePRK2 are localized to the plasma membrane/cell wall of growing pollen tubes. Both kinase domains have kinase activity when expressed in Escherichia coli. In phosphorylation assays with pollen membrane preparations, LePRK2, but not LePRK1, is phosphorylated, and the addition of tomato style, but not leaf, extracts to these membrane preparations results at least partially in specific dephosphorylation of LePRK2. Taken together, these results suggest that LePRK1 and LePRK2 play different roles in postpollination events and that at least LePRK2 may mediate some pistil response.     

肌醇磷脂信号途径参与胞外钙调素启动花粉萌发和花粉管伸长

高等植物有性生殖是植物发育生物学研究的重要内容之一,而作为雄配子体的花粉在雌蕊柱头上萌发及花粉管在花柱内的持续生长是有性生殖实现的关键。已有许多研究表明Ca2 在花粉萌发和花粉管生长过程中起重要作用。最近,我室在多年细胞外钙调素（calinodulin,CaM）存在。性质及生物学功能研究（孙大业等1995;Sun等1994,1995;Tang等1996）的基础上,通过不过膜的大分子CaM拈抗剂或抗体并结合恢复实验证实细胞外CaM对花粉的萌发和花粉管的伸长具有启动作用（马力耕和孙大业1996）,并发现G蛋白、质膜Caz”通道及胞内依赖Caz”的蛋白…     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser(277)/Ser(279)/Ser(282), resulted in longer pollen tubes, but alanine substitutions in motif II, Ser(304)/Ser(307)/Thr(308), resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status.     

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.     

Directional Guidance of Nicotiana alata Pollen Tubes in Vitro and on the Stigma

Pollen tubes navigate the route from stigma to ovule with great accuracy, but the cues that guide them along this route are not known. We reproduced the environment on the stigma of Nicotiana alata by immersing pollen in stigma exudate or oil close to an interface with an aqueous medium. The growth of pollen in this culture system mimicked growth on stigmas: pollen grains hydrated and germinated, and pollen tubes grew toward the aqueous medium. The rate-limiting step in pollen germination was the movement of water through the surrounding exudate or oil. By elimination of other potential guidance cues, we conclude that the directional supply of water probably determined the axis of polarity of pollen tubes and resulted in growth toward the interface. We propose that a gradient of water in exudate is a guidance cue for pollen tubes on the stigma and that the composition of the exudate must be such that it is permeable enough for pollen hydration to occur but not so permeable that the supply of water becomes nondirectional. Pollen tube penetration of the stigma may be the most frequently occurring hydrotropic response of higher plants.     

Plotting a course: multiple signals guide pollen tubes to their targets

Pollen plays a critical role in the life cycle of all flowering plants, generating a polarized pollen tube that delivers sperm to the eggs in the interior of the flower. Pollen tubes perceive multiple extracellular signals during their extended growth through different floral environments; these environments discriminate among pollen grains, allowing only those that are appropriately recognized to invade. The phases of pollen tube growth include interactions that establish pollen polarity, entry of pollen tubes into female cell walls, and adhesion-based pollen tube motility through a carbohydrate-rich matrix. Recent studies have identified cells within the female germ unit as important sources of pollen guidance cues. Other signals undoubtedly exist, and their discovery will require genetic screens that target diploid tissues as well as haploid male and female cells.     

Ca2+、pH在花粉及萌发花粉管生长中的作用研究进展

花粉正常萌发并生长是精细胞顺利到达胚囊并实现受精作用的前提,因而是高等植物有性生殖的一个关键环节。花粉管生长涉及一系列过程,而花粉(或花粉管)内外的Ca^2 和pH的变化与花粉萌发、花粉管生长有着密切的关系。比较详细地论述了Ca^2 和pH在花粉萌发、花粉管生长过程中的分布特点、生理功能及分子机制。