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.     

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.     

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.     

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     

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.     

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.     

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 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.     

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.     

New insights into pectin methylesterase structure and function

In bacteria, fungi and plants, pectin methylesterases are ubiquitous enzymes that modify the degree of methylesterification of pectins, which are major components of plant cell walls. Such changes in pectin structure are associated with changes in cellular adhesion, plasticity, pH and ionic contents of the cell wall and influence plant development and stress responses. In plants, pectin methylesterases belong to large multigene families, are regulated in a highly specific manner, and are involved in vegetative and reproductive processes, including wood and pollen formation, in addition to plant-pathogen interactions. Although, overall, protein structures are highly conserved between isoforms, recent data indicate that structural variations might be associated with the targeting and functions of specific pectin methylesterases.     

Hydrodynamics and cell volume oscillations in the pollen tube apical region are integral components of the biomechanics of Nicotiana tabacum pollen tube growth

Pollen tube growth is localized at the apex and displays oscillatory dynamics. It is thought that a balance between intracellular turgor pressure (hydrostatic pressure, reflected by the cell volume) and cell wall loosening is a critical factor driving pollen tube growth. We previously demonstrated that water flows freely into and out of the pollen tube apical region dependent on the extracellular osmotic potential, that cell volume changes reflect changes in the intracellular pressure, and that cell volume changes differentially induce, increases or decreases in specific phospholipid signals. This article shows that manipulation of the extracellular osmotic potential rapidly induces modulations in pollen tube growth rate frequencies, demonstrating that changes in the intracellular pressure are sufficient to reset the pollen tube growth oscillator. This indicates a direct link between intracellular hydrostatic pressure and pollen tube growth. Altering hydrodynamic flow through the pollen tube by replacing extracellular H₂O with ²H₂O adversely affects both cell volume and growth rate oscillations and induces aberrant morphologies. Normal growth and cell morphology are rescued by replacing ²H₂O with H₂O. Further studies revealed that the cell volume oscillates in the pollen tube apical region. These cell volume oscillations were not from changes in cell shape at the tip and were detectable up to 30 μm distal to the tip (the longest length measured). Cell volume in the apical region oscillates with the same frequency as growth rate oscillations but surprisingly the cycles are phase-shifted by 180°. Raman microscopy yields evidence that hydrodynamic flow out of the apex may be part of the biomechanics that drive cellular expansion. The combined results suggest that hydrodynamic loading/unloading in the apical region induces cell volume oscillations and has a role in driving cell elongation and pollen tube growth.     

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     

A Gain-of-Function Mutation of Arabidopsis Lipid Transfer Protein 5 Disturbs Pollen Tube Tip Growth and Fertilization

During compatible pollination of the angiosperms, pollen tubes grow in the pistil transmitting tract (TT) and are guided to the ovule for fertilization. Lily (Lilium longiflorum) stigma/style Cys-rich adhesin (SCA), a plant lipid transfer protein (LTP), is a small, secreted peptide involved in pollen tube adhesion-mediated guidance. Here, we used a reverse genetic approach to study biological roles of Arabidopsis thaliana LTP5, a SCA-like LTP. The T-DNA insertional gain-of-function mutant plant for LTP5 (ltp5-1) exhibited ballooned pollen tubes, delayed pollen tube growth, and decreased numbers of fertilized eggs. Our reciprocal cross-pollination study revealed that ltp5-1 results in both male and female partial sterility. RT-PCR and β-glucuronidase analyses showed that LTP5 is present in pollen and the pistil TT in low levels. Pollen-targeted overexpression of either ltp5-1 or wild-type LTP5 resulted in defects in polar tip growth of pollen tubes and thereby decreased seed set, suggesting that mutant ltp5-1 acts as a dominant-active form of wild-type LTP5 in pollen tube growth. The ltp5-1 protein has additional hydrophobic C-terminal sequences, compared with LTP5. In our structural homology/molecular dynamics modeling, Tyr-91 in ltp5-1, replacing Val-91 in LTP5, was predicted to interact with Arg-45 and Tyr-81, which are known to interact with a lipid ligand in maize (Zea mays) LTP. Thus, Arabidopsis LTP5 plays a significant role in reproduction.     

Arabidopsis COBRA‐LIKE 10, a GPI‐anchored protein,mediates directional growth of pollen tubes

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)‐anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C‐terminal hydrophobic residues. GPI‐anchored proteins are widespread cell sensors in mammals, especially during egg‐sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell‐cell communications in plants.     

Localization of pectins in the pollen tube wall of Ornithogalum virens L. Does the pattern of pectin distribution depend on the growth rate of the pollen tube?

Monoclonal antibodies that recognize pectins were used for the localization of esterified (JIM7) and acidic, unesterified (JIM5) forms of pectin in pollen tube walls of Ornithogalum virens L. (x = n = 3). The results indicated that the distribution of the two forms of pectin in the pollen tube wall depended on the medium (liquid or solid) used for pollen germination. In pollen tubes grown in the liquid medium, the localization of JIM7 was limited to the very tip of the pollen tube, whereas the localization of JIM5 indicated a uniform distribution of unesterified pectins in the very tip of the tube and along the subapical parts of the tube wall. In tubes germinated on the medium stabilized with agar (1–2%) the localization of JIM7 and JIM5 indicated the presence of both forms of pectin in the tube tip and along the whole length of the pollen tube wall in a ring-like pattern. Thus, the localization of esterified pectins in the sub-apical part of the pollen tube wall, below the apex of the tube, is described for the first time. Measurements of the growth rates of pollen tubes growing on the two types of medium indicated that oscillations in tube growth rate occur but these do not coincide with the pattern of pectin distribution in the tube wall. Our results complement the previous data obtained for the localization of JIM5 and JIM7 in pollen tube walls of other plant species. (Y.-Q. Li et al. 1994, Sex Plant Reprod 7: 145–150) and provide new insight into an understanding of the construction of the pollen tube wall and the physiology of pollen grain germination. Received: 25 January 1999 / Accepted: 23 June 1999     

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.     

Pollen-specific pectin methylesterase involved in pollen tube growth

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.     

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.