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.     

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     

Immunogold localization of arabinogalactan proteins,unesterified and esterified pectins in pollen grains and pollen tubes ofNicotiana tabacum L.

Summary The monoclonal antibodies JIM 5 (against unesterified pectin), JIM 7 (against methyl esterified pectin), MAC 207 (against arabinogalactan proteins, AGPs), and JIM 8 (against a subset of AGPs) were utilized singly or in combinations for immunogold labelling of germinated pollen grains and pollen tubes ofNicotiana tabacum. Pectins were localized in the inline of pollen grain, unesterified pectin being more abundant than the esterified one. AGPs were co-localized with pectin in the inline, but were present preferably close to the plasma membrane. In pollen tubes, AGPs, unesterified and esterified pectins were co-localized in the outer and middle layers of the cell wall. The density of the epitopes was not uniform along the length of the pollen tube, but showed alterations. In the pollen tube tip wall esterified pectin was abundantly present, but not AGPs. In the cytoplasm esterified pectin and AGPs were detected in Golgi derived vesicles, indicating their role in the pathway of the cell wall precursors. In the cell wall of generative cell only AGPs, but no pectins were localized. The co-localization of pectins and AGPs in the cell wall of pollen grain and pollen tube might play an important role, not only in maintenance of the cell shape, but also in cell-cell interaction during pollen tube growth and development.Abbreviations AGP arabinogalactan protein - BSA bovine serum albumin - GA glutaraldehyde - MAb monoclonal antibody - NGS normal goat serum - PFA paraformaldehyde     

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.     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

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     

Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside

Arabinogalactan proteins (AGPs) are abundant complex macromolecules involved in both reproductive and vegetative plant growth. They are secreted at pollen tube tips in Lilium longiflorum. Here, we report the effect of the (beta-D-glucosyl)3 Yariv phenylglycoside, known to interact with AGPs, on pollen tube extension in several plant species. In Annona cherimola the Yariv reagent clearly inhibited pollen tube extension within 1-2 h of treatment, as demonstrated previously for L. longiflorum, but had no effect on Lycopersicon pimpinellifolium, Aquilegia eximia, and Nicotiana tabacum. With the monoclonal antibody JIM13 we also examined these same species for evidence that they secreted AGPs at their pollen tube tips. Only A. cherimola showed evidence of AGPs at the pollen tube tip as does lily. The Yariv reagent causes arrest of tube growth in both A. cherimola and lily, but its removal from the medium allows regeneration of new tip growth in both species. We show that the site of the new emerging tip in lily can be predicted by localization of AGP secretion. Labeling with JIM13 appeared on the flanks of the arrested tip 1 h after removal of the Yariv reagent from the growth medium. After 4 h, many of the Yariv reagent-treated pollen tubes had regenerated new pollen tubes with the tips brightly labeled by JIM13 and with a collar of AGPs left at the emergence site. During this recovery, esterified pectins colocalized with AGPs. Secretion at the site of the new tip may be important in the initial polarization event that occurs on the flanks of the arrested tube tip and results in a new pollen tube.     

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.     

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     

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 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     

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.     

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.     

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.     

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     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

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.     

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.