A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix

Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis.     

Adhesion of lily pollen tubes on an artificial matrix

 We proposed that pollination in lily is a case of cell adhesion and cell movement, but experimental evidence for the adhesion event is lacking. In this study, we developed an artificial extracellular matrix that mimics the in vivo lily stylar transmitting tract. This artificial matrix was created by applying the transmitting tract exudate extracted from lily styles onto a nitrocellulose membrane. When in vitro-grown pollen tubes were applied to the matrix, they adhered by their tips to the area of the stylar exudate which is rich in arabinogalactan proteins. Once they adhered, they grew on the in vitro artificial matrix at rates faster than normal. This is the first experimental evidence demonstrating the adhesion of in vitro-grown pollen tubes, an event that has been described as common in vivo. The adhesion event is stylar exudate specific, concentration dependent, and is affected by the developmental age of the pollen tube. This bioassay for pollen tube adhesion will be used to isolate the adhesive molecules from the stylar exudate. Received: 9 December 1996 / Revision accepted: 5 May 1997     

Expression studies of SCA in lily and confirmation of its role in pollen tube adhesion

During pollination the pollen tube grows into the style and toward the ovary via the transmitting tract. In lily the growth of pollen tubes involves tube cell adhesion to transmitting tract cells. We reported two molecules involved in this adhesion event. One is a pectic polysaccharide and the other, a 9 kDa basic protein named SCA for stigma/stylar cysteine-rich adhesin. SCA, which shows some identity with LTP (lipid transfer protein), was localized to the transmitting tract epidermis of the style where pollen tubes adhere. The present studies on the expression of SCA indicate that the protein has a similar expression pattern with LTP1 in Arabidopsis and that the protein is abundant in both the stigma and the style. For further proof of its role in pollen tube adhesion the activity of Escherichia coli-expressed protein has been studied in an in vitro adhesion assay system.     

Lily (<Emphasis Type="Italic">Lilium longiflorum</Emphasis> L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.     

A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix

Pollen tube cells adhere to the wall surface of the stylar transmitting tract epidermis in lily. This adhesion has been proposed as essential for the proper delivery of the sperm cells to the ovule. An in vitro adhesion bioassay has been used to isolate two stylar molecules required for lily pollen tube adhesion. The first molecule was determined to be a small, cysteine-rich protein with some sequence similarity to lipid transfer proteins and now called stigma/stylar cysteine-rich adhesin (SCA). The second, larger, molecule has now been purified from style fragments and characterized. Chemical composition, specific enzyme degradations, and immunolabeling data support the idea that this molecule required for pollen tube adhesion is a pectic polysaccharide. In vitro binding assays revealed that this lily stylar adhesive pectin and SCA are able to bind to each other in a pH-dependent manner.     

Adhesion molecules in lily pollination

Adhesion occurs both between pollen tubes and between the pollen tube and transmitting tract epidermis (TTE) in lily. The stylar matrix secreted by the TTE can be isolated and used in an in vitro adhesion assay for pollen tubes. This bioassay was used to isolate two stigma/stylar adhesion molecules in lily: a pectic polysaccharide and a small cysteine-rich, basic protein we named SCA (stigma/stylar cysteine-rich adhesin). Both molecules were purified and used in an adhesion assay. Adhesion in the assay can be disrupted by treatment of the pectin with polygalacturonase and of SCA with proteinase K. The two molecules bind to each other in a pH-dependent fashion, and this binding is necessary for the adhesion assay to work. Antibodies to each of the molecules show their localization at the sites of pollen tube adhesion in the style. Pollen does not produce SCA but does bind this protein in vivo and in vitro. In vivo functional analyses are necessary to establish the roles of these molecules in lily pollination. Received: 29 October 2000 / Accepted: 17 April 2001     

Adhesion and guidance in compatible pollination

The mechanisms of compatible pollination are less studied than those of incompatible pollination and yet most of the angiosperms show self-compatibility. From the release of pollen from anthers to the penetration of the micropyle by the pollen tube tip, there are numerous steps where the interaction between pollen and the pistil can be regulated. Recent studies have documented some diverse ways in which pollen tubes carrying sperm cells are guided to the ovules through the pistil extracellular matrices of the transmitting tract. What is still missing is an understanding of pollen tube cell biology in vivo. A recent finding supports the role of the synergids in the crucial guidance cue for the pollen tube tip at the micropyle, but experimental evidence for other 'guidepost' cells in the pistil is still lacking. The fact that the pollen tube must first travel through the matrices of the stigma and style before it can respond to the cue from the ovule makes it likely that there is a hierarchy of signalling events in pollen-pistil interactions starting at the stigma and ending at the micropyle. On the pistil side, several model systems have been used in the discovery of molecules implicated in either physical or chemical guidance. In lily, which has a hollow style, adhesion molecules (pectin and SCA) are implicated in guidance. SCA alone is also capable of inducing pollen chemotropism in an in vitro assay, suggesting that this peptide plays a dual role in lily pollination: chemotactic in the stigma and haptotactic (adhesion mediated) in the style.     

Immunocytochemical localization of esterified and unesterified pectins in unpollinated and pollinated styles of Petunia hybrida Hort

Localization of pectins in the style of Petunia hybrida before and after pollination was investigated by immunocytochemistry using two primary monoclonal antibodies specific to highly (JIM7) and weakly (JIM5) methylesterified pectins. In the unpollinated style, esterified pectins occurred mainly in the cell walls of cortex tissue, while unesterified pectins were present mainly in the extracellular matrix (ECM) of the transmitting tract. After pollination no remarkable differences were found in pectin distribution in the ground tissue of the style. On the other hand, in the transmitting tract a reduction in the quantity of unesterified pectins was observed. Unesterified pectins in the extracellular regions of the transmitting tissue decreased before the penetration of the pollen tubes, indicating that pollination induces a reduction in the amount of unesterified pectins in the transmitting-tract ECM. The correlation between the degradation of strongly Ca2+-binding pectins and the growing level of those ions in the extracellular regions of the transmitting tract in the pollinated pistil of P. hybrida (M. Lenartowska et al. 1997) suggests that this process may constitute a mechanism for creating an optimum calcium medium for in vivo-growing pollen tubes. Both pectin categories were localized in pollen tubes. Esterified pectin epitopes were localized mainly in the vesicles of the tip cytoplasm. Unesterified pectin epitopes were found in the external fibrillar wall of pollen tubes.     

Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination

In lily, adhesion of the pollen tube to the transmitting-tract epidermal cells (TTEs) is purported to facilitate the effective movement of the tube cell to the ovary. In this study, we examine the components of the extracellular matrices (ECMs) of the lily pollen tubes and TTEs that may be involved in this adhesion event. Several monoclonal antibodies to plant cell wall components such as esterified pectins, unesterified pectins, and arabinogalactan-proteins (AGPs) were used to localize these molecules in the lily pollen tube and style at both light microscope (LM) and transmission electron microscope (TEM) levels. In addition, (-d-Glc)₃ Yariv reagent which binds to AGPs was used to detect AGPs in the pollen tube and style. At the LM level, unesterified pectins were localized to the entire wall in in-vivo- and in-vitro-grown pollen tubes as well as to the surface of the stylar TTEs. Esterified pectins occurred at the tube tip region (with some differences in extent in in-vivo versus in-vitro tubes) and were evenly distributed in the entire style. At the TEM level, esterified pectins were detected inside pollen tube cell vesicles and unesterified pectins were localized to the pollen tube wall. The in-vivo pollen tubes adhere to each other and can be separated by pectinase treatment. At the LM level, AGP localization occurred in the tube tip of both in-vivo- and in-vitro-grown pollen tubes and, in the case of one AGP probe, on the surface of the TTEs. Another AGP probe localized to every cell of the style except the surface of the TTE. At the TEM level, AGPs were mainly found on the plasma membrane and vesicle membranes of in-vivo-grown pollen tubes as well as on the TTE surface, with some localization to the adhesion zone between pollen tubes and style. (-d-Glc)₃ Yariv reagent bound to the in-vitro-grown pollen tube tip and significantly reduced the growth of both in-vitro- and in-vivo-grown pollen tubes. This led to abnormal expansion of the tube tip and random deposition of callose. These effects could be overcome by removal of (-d-Glc)₃ Yariv reagent which resulted in new tube tip growth zones emerging from the flanks of the arrested tube tip. The possible roles of pectins and AGPs in adhesion during pollination and pollen tube growth are discussed.Abbreviations AGP arabinogalactan-protein - ECM extracellular matrix - Glc glucose - MAbs monoclonal antibodies - LM light microscope - Man mannose - TEM transmission electron microscope - TTE transmitting tract epidermal cell The authors thank Michael Georgiady for assistance with the preparation of material for the TEM immunolocalization, Diana Dang for her help with the pectinase experiment, and Kathleen Eckard for assistance in all aspects of this study. The MAbs were the generous gifts of Dr. J.P. Knox. G.Y. Jauh thanks Dr. E.A. Nothnagel for assistance in making the Yariv reagent and for the gift of the control (-d-Man)₃ Yariv reagent. This work is in partial fulfilment of the dissertation requirements for a PhD degree in Botany and Plant Sciences for G.Y. Jauh at the University of California, Riverside. This work was supported by National Science Foundation grant 91-18554 and an R.E.U. grant to E.M.L.     

Calcium snapshots in the stigma and style of medlar (Lycium barbarum L.) during pollen germination and pollen tube growth suggest active calcium oscilations

Abstract

The stigma (tip of the pistil) of medlar is wet and covered with stigmatic exudate at anthesis. The exudate contains many vesicles with abundant calcium precipitates. After deposition on the stigma, the pollen grain undergoes hydration, displaying signs of calcium ion (Ca²⁺) transfer from the exudate vesicles into the pollen grains. Calcium precipitates in the pollen cytoplasm are concentrated into small vacuoles that fuse to form large vacuoles, which provide turgor pressure to push the cytoplasm to the apical region of the growing pollen tube. Many calcium precipitates are present in the stylar transmitting tract, which displays a calcium gradient: fewer precipitates are localised in the distal (upper) transmitting tissue below the stigma, and more precipitates are present in the transmitting tract at the style base. The emporal and spatial distribution of calcium in the stigma and style of medlar suggests that it satisfies the demand for calcium in vivo and played some functional significance.     

Pollen tube growth and guidance: roles of small, secreted proteins

BACKGROUND: Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen-pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization. SCOPE: In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin.     

VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.     

烟草花粉萌发和花粉管生长期间柱头和花柱中的钙分布

烟草柱头表面有两层覆盖物,其中含有少量细小的钙颗粒.花粉落到柱头上后,储存在花粉外壁中的钙被释放到覆盖层中.当花粉管穿过覆盖层长入柱头细胞之间时,花粉管顶端的细胞壁中出现了大量的细小钙颗粒.开花后22 h观察时,在花柱引导组织中形成了钙的梯度分布:花柱上部引导组织中的钙较少,而下部连接子房处的花柱引导组织中含有较多的钙颗粒.去雄花开花后1 d时,花柱上部引导组织中的钙明显增多;3 d时,连柱头细胞中也出现了较多的钙颗粒.讨论了烟草花柱引导组织中钙梯度分布和花粉管生长的关系.     

Pollen tube growth in association with a dry-type stigmatic transmitting tissue and extragynoecial compitum in the basal angiosperm Kadsura longipedunculata (Schisandraceae)

Spatial features of pollen tube growth and the composition of the extracellular matrix (ECM) of transmitting tissue in carpels of Kadsura longipedunculata, a member of the basal angiosperm taxon Schisandraceae, were characterized to identify features of transmitting tissue that might have been important for pollen-carpel interactions during the early history of angiosperms. In addition to growing extracellularly along epidermal cells that make up stigmatic crests of individual carpels, pollen tubes grow on abaxial carpel epidermal cells between unfused carpels along an extragynoecial compitum to subsequently enter an adjacent carpel, a feature important for enhancing seed set in apocarpous species. Histo- and immunochemical data indicated that transmitting tissue ECM is not freely flowing as previously hypothesized. Rather, the ECM is similar to that of a dry-type stigma whereby a cuticular boundary with associated esterase activity confines a matrix containing methyl-esterified homogalacturonans. The Schisandraceae joins an increasing number of basal angiosperm taxa that have a transmitting tissue ECM similar to a dry-type stigma, thereby challenging traditional views that the ancestral pollen tube pathway was similar to a wet-type stigma covered with a freely flowing exudate. Dry-type stigmas are posited to provide tighter control over pollen capture, retention, and germination than wet-type stigmas.     

A Homolog of the Substrate Adhesion Molecule Vitronectin Occurs in Four Species of Flowering Plants

The extracellular matrix (ECM) has been implicated in the primary developmental processes of many organisms. A family of secretory adhesive glycoproteins called substrate adhesion molecules (SAMs) is believed to confer these dynamic capabilities to the ECM in animals. In this paper, we report the existence of SAM-like genes and gene products in flowering plants. Hybridizations with a human vitronectin cDNA probe and genomic DNA from broad bean, soybean, and tomato revealed vitronectin-like sequences. Human vitronectin antibodies cross-react with a 55-kilodalton protein in leaf and root protein extracts from lily, broad bean, soybean, and tomato. In addition, immunocytochemical staining of frozen sections of lily leaf and broad bean gynoecium demonstrated that vitronectin-like proteins were localized to the ECM on the cell surface, with the most intense labeling residing in the transmitting tract of broad bean gynoecium.     

Plantacyanin plays a role in reproduction in Arabidopsis

Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-beta-glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wild-type pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.     

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.