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 and cell movement during pollination: cherchez la femme

Pollination involves an interaction between the female tissues (stigma, style and ovary) and the male gametophyte or the pollen tube cell, which contains the sperm cells. Freezing methods now allow us to visualize the extracellular matrices that guide pollen tubes to the ovary. Adhesion of the pollen tube to these specialized extracellular matrices might be a mechanism of guidance and tube cell movement in the style. In lily, the stylar adhesion molecules are a pectin and a small, basic cysteine-rich protein, both of which are necessary to induce tube cell adhesion to an artificial, in vitro style matrix.     

Exogenous free ubiquitin enhances lily pollen tube adhesion to an in vitro stylar matrix and may facilitate endocytosis of SCA

Pollen tube adhesion and guidance on extracellular matrices within the pistil are essential processes that convey the pollen tube cell and the sperm cells to the ovule. In this study, we purified an additional molecule from the pistil that enhances pollen tube adhesion when combined with the SCA (stigma/stylar cysteine-rich adhesin)/pectin matrix in our in vitro assay. The enhancer of adhesion was identified as free ubiquitin (Ub). This was confirmed by use of bovine Ub as a substitute for lily (Lilium longiflorum Thunb.) stigma Ub. To study the interaction of SCA and Ub with the lily pollen tube, we labeled both proteins with biotin. We observed uptake of biotin-labeled SCA and Ub into the pollen tube cells in vitro using confocal microscopy. For SCA, a strong signal occurred first at the tip of the pollen tube, suggestive of an endocytosis event, and then progressively throughout the tube cytoplasm. SCA was also localized inside the in vivo pollen tube using immunogold electron microscopy and found to be present in endosomes, multivesicular bodies, and vacuoles, all known to be endocytic compartments. It was also confirmed that SCA is endocytosed in the in vitro adhesion assay. Internalization of SCA was increased in pollen tubes treated with exogenous Ub compared to those without Ub, suggesting that Ub may facilitate SCA endocytosis. These results show that Ub can act as an enhancer of pollen tube adhesion in vitro and that it is taken up into the pollen tube as is SCA. The Ub machinery may play a role in pollen tube adhesion and guidance in lily.     

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 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.     

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.     

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     

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.     

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.     

Cell surface arabinogalactan-proteins and their relation to cell proliferation and viability

Summary We have used high-pressure freezing followed by freeze substitution (HPF/FS) to preserve in vivo grown lily pollen tubes isolated from the style. The results indicated that HPF/FS (i) allows excellent preservation of the pollen tubes, (ii) maintains in situ the stylar matrix secreted by the transmitting tract cells, and (iii) preserves the interactions that exist between pollen tubes. Particular attention has been given to the structure of the pollen tube cell wall and the zone of adhesion. The cell wall is composed of an outer fibrillar layer and an inner layer of material similar in texture and nature to the stylar matrix and that is not callose. The stylar matrix labels strongly for arabinogalactan proteins (AGPs) recognized by monoclonal antibody JIM13. The zone of adhesion between pollen tubes contains distinct matrix components that are not recognized by JIM13, and apparent cross-links between the two cell walls. This study indicates that HPF/FS can be used successfully to preserve in vivo grown pollen tubes for ultrastructural investigations as well as characterization of the interactions between pollen tubes and the stylar matrix.Abbreviations AGPs arabinogalactan proteins - FS freeze substitution - HPF high-pressure freezing     

Two SCA (stigma/style cysteine-rich adhesin) isoforms show structural differences that correlate with their levels of in vitro pollen tube adhesion activity

Lily pollen tubes grow adhering to an extracellular matrix produced by the transmitting tract epidermis in a hollow style. SCA, a small ( approximately 9.4 kDa), basic protein plus low esterified pectin from this extracellular matrix are involved in the pollen tube adhesion event. The mode of action for this adhesion event is unknown. We partially separated three SCA isoforms from the lily stigma in serial size exclusion column fractions (SCA1, 9370 Da; SCA2, 9384 Da; SCA3, 9484 Da). Peptide sequencing analysis allowed us to determine two amino acid variations in SCA3, compared with SCA1. For SCA2, however, there are more sequence variations yet to be identified. Our structural homology and molecular dynamics modeling results show that SCA isoforms have the plant nonspecific lipid transfer protein-like structure: a globular shape of the orthogonal 4-helix bundle architecture, four disulfide bonds, an internal hydrophobic and solvent-inaccessible cavity, and a long C-terminal tail. The Ala(71) in SCA3, replacing the Gly(71) in SCA1, has no predictable effect on structure. The Arg(26) in SCA3, replacing the Gly(26) in SCA1, is predicted to cause structural changes that result in a significantly reduced volume for the internal hydrophobic cavity in SCA3. The volume of the internal cavity fluctuates slightly during the molecular dynamics simulation, but overall, SCA1 displays a larger cavity than SCA3. SCA1 displays higher activity than SCA3 in the in vitro pollen tube adhesion assay. No differences were found between the two SCAs in a binding assay with pectin. The larger size of the hydrophobic cavity in SCA1 correlates with its higher adhesion activity.     

A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.     

Transmitting tissue ECM distribution and composition, and pollen germinability in Sarcandra glabra and Chloranthus japonicus (Chloranthaceae)

BACKGROUND: and Aims Free-flowing surface exudates at the stigmatic (wet versus dry stigma) and adaxial epidermis at the site of angiospermy in carpels of Chloranthaceous species have been proposed to comprise a continuous extracellular matrix (ECM) operating in pollen tube transmission to the ovary. The aim of this research was to establish the spatial distribution and histo/immunochemical composition of the ECM involved in pollen tube growth in Sarcandra glabra and Chloranthus japonicus (Chloranthaceae). METHODS: Following confirmation of the pollen tube pathway, the histo/immunochemical make-up of the ECM was determined with histochemistry on fresh tissue to detect cuticle, esterase, proteins, pectins, and lipids and immunolocalization at the level of the TEM on sections from cryofixed/freeze-substituted tissue to detect molecules recognized by antibodies to homogalacturonans (JIM7, 5), arabinogalactan-proteins (JIM13) and cysteine-rich adhesion (SCA). KEY RESULTS: Pollen germinability is low in both species. When grains germinate, they do so on an ECM comprised of an esterase-positive cuticle proper (dry versus wet stigma). Pollen tubes do not track the surface ECM of stigma or adaxial epidermal cells at the site of angiospermy. Instead, tubes grow between stigmatic cells and subsequently along the inner tangential walls of the stigmatic and adaxial carpel cells at the site of angiospermy. Pollen tubes enter the ovary locule at the base of the funiculus. The stigmatic ECM is distinct by virtue of the presence of anti-JIM5 aggregates, lipids, and a protein recognized by anti-SCA. CONCLUSIONS: The Chloranthaceae joins a growing number of basal angiosperm taxa whereby pollen tubes germinate on a dry versus wet stigma to subsequently grow intercellularly en route to the ovary thereby challenging traditional views that the archetype pollen tube pathway was composed of the surface of stigma and adaxial epidermal cells covered with a free-flowing exudate.     

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.     

Stylar glycoproteins bind to S-RNase in vitro

S-RNases determine the specificity of S-specific pollen rejection in self-incompatible plants of the Solanaceae, Rosaceae, and Scrophulariaceae. They are also implicated in at least two distinct types of unilateral interspecific incompatibility in Nicotiana. However, S-RNase itself is not sufficient for most types of pollen rejection, and evidence for its direct interaction with pollen tubes is limited. Thus, non-S-RNase factors also are required for pollen rejection. As one approach to identifying such factors, we tested whether SC10-RNase from Nicotiana alata would bind to other stylar proteins in vitro. SC10-RNase was immobilized on Affi-gel, and binding proteins were analyzed by SDS-PAGE and immunoblotting. In addition to SC10-RNase and a small protein similar to lily chemocyanin, the most prominent binding proteins include NaTTS, 120K, and NaPELPIII, these latter three being arabinogalactan proteins previously shown to interact directly with pollen tubes. We also show that SC10-RNase and these glycoproteins migrate as a complex in a native PAGE system. Our hypothesis is that S-RNase forms a complex with these glycoproteins in the stylar ECM, that the glycoproteins interact directly with the pollen tubes and thus that the initial interaction between the pollen tube and S-RNase is indirect.     

Integrin-like proteins in the pollen tube: detection, localization and function

The distribution of integrin-like proteins in the pollen tube was examined by immunofluorescent labeling and western blotting techniques using antibodies against human placenta integrin vitronectin receptor (VnR), and alpha(v), beta3 and beta1 integrin subunits. Pseudocolor-coded confocal images showed intense immunostaining within 10 and 5 microm of the tip of the pollen tube in Lilium davidii and Nicotiana tabacum respectively. In both segments the site near the plasma membrane was labeled. Western blotting analyses revealed cross-reaction of anti-beta3, anti-alpha(v) and anti-VnR with the proteins in the plasma membrane preparation of L. davidii and Hemerocallis citrina pollen tube. These studies provide evidence for the first time that the integrin-like protein is present in pollen tubes, and it may be mainly composed of alpha(v) and beta3 subunits in lily pollen tubes. In a functional assay, neither anti-VnR antibody nor the Arg-Gly-Asp-Ser tetrapeptide inhibited pollen tube growth of N. tabacum in vitro, but both of them depressed tube growth on the stigma and in style under quasi in vivo culture conditions. The integrin-like proteins localized in the tip and periphery of the pollen tube appeared to play roles in growth of the pollen tube tip and interaction with the extracellular matrix of the style.     

Calcium levels increase in the lily stylar transmitting tract after pollination

Pollen tube growth in vitro requires calcium for most species but the in vivo source or reservoir of this calcium is not known. Using methods to localize calcium in situ, we confirm that low levels of calcium are detected in the transmitting tract extracellular matrix (ECM) in unpollinated lily styles. Pollination in lily induces an increase in the detectable levels of calcium in the transmitting tract ECM binding to the stylar cell and pollen tube walls. This calcium is detected in the cytoplasm and vesicles near the pollen tube tip.An erratum to this article can be found at     

Movement of the tube cell in the lily style in the absence of the pollen grain and the spent pollen tube

Our model proposes that pollen tube growth is a form of cell movement where the tube tip can be considered analogous to a migrating cell which leaves a trail of extracellular matrix (the spent pollen tube) behind. We demonstrate that the tube cell can convey the sperm cells to the ovule and effect fertilization even in the absence of the pollen grain and the spent pollen tube. Adhesion is an integral part of cell attachment and movement in animal systems. We show that in vivo-grown pollen tubes grow beneath the cuticle of the stylar transmitting tract epidermis and directly adhere to one another and the outer wall of the epidermal cells. A fibrous wall material is found to cover the tip of the pollen tube cell wall and the surface of the transmitting tract cells where the two adhere. Fixation methods to preserve adhesive compounds were used. The pollen-tubes grown in vivo, but not in vitro, show star-shaped clusters of F-actin microfilaments in the region back from the tip, as seen by rhodamine-phalloidin staining. These configurations are similar to focal adhesions seen in moving animal cells.     

Pollen-stigma adhesion in Arabidopsis: a species-specific interaction mediated by lipophilic molecules in the pollen exine.

To investigate the nature and role of cell adhesion in plants, we analyzed the initial step of pollination in Arabidopsis: the binding of pollen grains to female stigma cells. Here we show this interaction occurs within seconds of pollination. Because it takes place prior to pollen hydration, it also requires adhesion molecules that can act in a virtually dry environment. We developed assays that monitored adhesion of populations of pollen grains and individual cells. Adhesion between pollen and stigma cells is highly selective - Arabidopsis pollen binds with high affinity to Arabidopsis stigmas, while pollen from other species fails to adhere. Initial binding is independent of the extracellular pollen coat (tryphine), indicating that adhesion molecules reside elsewhere on the pollen surface, most likely within the exine walls. Immediately after pollination, the stigma surface becomes altered at the interface, acquiring a pattern that interlocks with the exine; this pattern is evident only with pollen from Arabidopsis and its close relatives. Purified exine fragments bind to stigma cells, and biochemical analyses indicate that this specific, rapid and anhydrous adhesion event is mediated by lipophilic interactions.