Arabinogalactan Proteins Research Articles

In vitro characterization of root extracellular trap and exudates of three Sahelian woody plant species.

Arabinogalactan protein content in both root extracellular trap and root exudates varies in three Sahelian woody plant species that are differentially tolerant to drought. At the root tip, mature root cap cells, mainly border cells (BCs)/border-like cells (BLCs) and their associated mucilage, form a web-like structure known as the "Root Extracellular Trap" (RET). Although the RET along with the entire suite of root exudates are known to influence rhizosphere function, their features in woody species is poorly documented. Here, RET and root exudates were analyzed from three Sahelian woody species with contrasted sensitivity to drought stress (Balanites aegyptiaca, Acacia raddiana and Tamarindus indica) and that have been selected for reforestation along the African Great Green Wall in northern Senegal. Optical and transmission electron microscopy show that Balanites aegyptiaca, the most drought-tolerant species, produces only BC, whereas Acacia raddiana and Tamarindus indica release both BCs and BLCs. Biochemical analyses reveal that RET and root exudates of Balanites aegyptiaca and Acacia raddiana contain significantly more abundant arabinogalactan proteins (AGPs) compared to Tamarindus indica, the most drought-sensitive species. Root exudates of the three woody species also differentially impact the plant soil beneficial bacteria Azospirillum brasilense growth. These results highlight the importance of root secretions for woody species survival under dry conditions.

Immunocytochemical studies on the distribution of arabinogalactan proteins (AGPs) as a response to fungal infection in Malus x domestica fruit

Arabinogalactan proteins (AGPs) are cell components implicated in plant-microbe interactions. Despite the significance of AGPs in response to stress factors, their distribution during development of fungal disease in fruit is unknown. In our work, in situ analysis of AGP arrangement in fruit inoculated with Penicillium spinulosum during the consecutive days of infection development was carried out. For immunolocalization of AGPs, samples were incubated with JIM13, MAC207, LM2, and LM14 antibodies recognizing the AGP carbohydrate moieties. To analyse cell walls without proper action of AGP, an experiment with β-glucosyl Yariv reagent specifically binding AGPs was performed. The results showed an increase of signal fluorescence in the fruit after 16 days of fungal disease. Higher amounts of the examined epitopes were observed in the infection-altered sites of the fruit, in close vicinity to a surface filled by fungal spores. The results indicate that the Yariv reagent treatment induced progress of the fungal disease. Changes in the AGP presence during the fungal disease confirmed their involvement in defence against pathogen attack in fruit.

Inhibition Mechanisms of Wine Polysaccharides on Salivary Protein Precipitation

In this work, high-performance liquid chromatography, fluorescence quenching, nephelometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis were used to study the effect of polysaccharides naturally present in wine [rhamnogalacturonan II (RG II) and arabinogalactan proteins (AGPs)] on the interaction between salivary proteins (SP) together present in saliva and tannins (punicalagin (PNG) and procyanidin B2). In general, the RG II fraction was more efficient to inhibit SP precipitation by tannins, especially for acidic proline-rich proteins (aPRPs) and statherin/P-B peptide, than AGPs. The RG II fraction can act mainly by a competition mechanism in which polysaccharides compete by tannin binding. However, in the presence of Na+ ions in solution, no RG II effect was observed on SP-tannin interactions. On the other hand, dependent upon the saliva sample as well as the tannin studied, AGPs can act by both mechanisms, competition and ternary (formation of a ternary complex with SP-tannin aggregates enhancing their solubility).

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Aluminum toxicity-induced pollen tube growth inhibition in apple (Malus domestica) is mediated by interrupting calcium dynamics and modification of cell wall components

Aluminum (Al) is known the most abundant metal in the Earth’s crust which is a major problem limiting crop production and yield throughout the world. Al could inhibit pollen tube elongation, however the mechanism is largely unknown. The aim of this study was to investigate the influence of Al toxicity on Malus domestica pollen tube growth and possible cytological mechanism. The results showed that Al inhibited pollen germination and tube growth in a concentration dependent manner. An non-invasive ion-selective electrode technique together with fluorescent labeling indicated that Al toxicity decreased pollen tube apex calcium influx and disrupted [Ca2+]c gradient. Al toxicity also altered actin filament orientation. Results from immuno-localization and fluorescence labeling indicated that Al toxicity affected the accumulation and distribution of callose, acid pectins, esterified pectins and arabinogalactan proteins in pollen tubes, which were supported by Fourier-transform infrared analysis. In a word, Al induced a series of the cellular toxic changes concerning calcium influx, [Ca2+]c gradient, actin and cell wall components. Based on the information provided in this article, it is concluded that Al toxicity inhibits apical growth of pollen tubes by changing calcium influx and [Ca2+]c and cell wall components.

Changes in the Soluble and Insoluble Compounds of Shelf-Stable Orange Juice in Relation to Non-Enzymatic Browning during Storage.

For the first time in literature, this study revealed the participation of polymeric components of orange juice cloud and pulp (such as proteins, arabinogalactan proteins, or protein-pectin complexes) during nonenzymatic browning. In a quest to better understand the nonenzymatic browning of shelf-stable orange juice during storage, the juice was fractionated into different fractions depending on the solubility in water/ethanol and the obtained fractions were characterized. The results showed that brown compounds that were formed during storage of orange juice were distributed over water insoluble (pulp), ethanol insoluble (cloud), and ethanol soluble (serum) fractions. In the ethanol insoluble fraction, the brown compounds are hypothesized to be associated with proteins, arabinogalactan proteins, and/or protein-pectin complexes of this fraction without significantly changing their molecular weight distributions, monosaccharide compositions, and protein contents. The changes in the ethanol soluble fraction including ascorbic acid degradation, acid-catalyzed hydrolysis of sucrose, and formation of furfural and 5-hydroxymethylfurfural were highly correlated to the browning development of the juice during storage.

Galling insects as phenotype manipulators of cell wall composition during the development of galls induced on leaves of Aspidosperma tomentosum (Apocynaceae)

The gall structure is built from redifferentiation of host plant tissues, changing the cell wall proprieties and associated functions. Because of the oxidative stress imposed by the galling insect on the plant tissues, some phenotypic traits associated with cell wall metabolism may be suppressed during gall development. Using an immunocytochemical approach we investigated the pectin and glycoprotein composition of the cell walls during non-galled leaf development and galls induced on Aspidosperma tomentosum (Apocynaceae). Embedding in historesin was used for anatomical characterization, as well as for immunocytochemical analyses. The samples were probed with the primary monoclonal antibodies JIM5, JIM7, LM1, LM2, LM5 and LM6. The gall develops especially from cell hypertrophy and hyperplasia of the spongy and palisade parenchyma, which determine its shape. Epitopes of β-D-galactan and α-L-arabinan in the galls were totally suppressed or less expressed. The increased labeling of homogalacturonan epitopes, especially in senescent galls, is a clear indicator that pectin methylesterase activity is maintained despite oxidative stresses imposed by the galling insect. Arabinogalactan proteins were not detected in any non-galled leaf or gall stages. The galling insect deeply changed the structure and deposition pattern of the cell wall, promoting new functionalities in the gall tissue compartments.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with β-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked β-galactosyl backbone chains substituted with side chains of (1 → 6)-linked β-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-β-(1 → 3)-galactanase with the aid of endo-β-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of β-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked β-galactosyl side chains of varying length along the (1 → 3)-linked β-galactosyl backbone chains.

Ragp: Pipeline for mining of plant hydroxyproline-rich glycoproteins with implementation in R.

Hydroxyproline-rich glycoproteins (HRGPs) are one of the most complex families of macromolecules found in plants, due to the diversity of glycans decorating the protein backbone, as well as the heterogeneity of the protein backbones. While this diversity is responsible for a wide array of physiological functions associated with HRGPs, it hinders attempts for homology based identification. Current approaches, based on identifying sequences with characteristic motifs and biased amino acid composition, are limited to prototypical sequences. Ragp is an R package for mining and analysis of HRGPs, with emphasis on arabinogalactan proteins. The ragp filtering pipeline exploits one of the HRGPs key features, the presence of hydroxyprolines which represent glycosylation sites. Main package features include prediction of proline hydroxylation sites, amino acid motif and bias analyses, efficient communication with web servers for prediction of N-terminal signal peptides, glycosylphosphatidylinositol modification sites and disordered regions and the ability to annotate sequences through hmmscan and subsequent GO enrichment, based on predicted Pfam domains. As such, ragp extends R's rich ecosystem for high-throughput sequence data analyses. The ragp R package is available under the MIT Open Source license and is freely available to download from GitHub at: https://github.com/missuse/ragp.

In silico and expression analyses of fasciclin-like arabinogalactan proteins reveal functional conservation during embryo and seed development

Key messageThe fasciclin-like arabinogalactan proteins organization into four groups is conserved and may be related to specific roles in developmental processes across angiosperms.Fasciclin-like arabinogalactan proteins (FLAs) are a subclass of arabinogalactan proteins (AGPs), which contain fasciclin-like domains in addition to typical AGP domains. FLAs are present across all embryophytes, and despite their low overall sequence similarity, conserved regions that define the fasciclin functional domain (FAS) have been identified, suggesting that the cell adhesion property is also conserved. FLAs in Arabidopsis have been organized into four subgroups according to the number and distribution of functional domains. Recent studies associated FLAs with cell wall-related processes where domain organization seemed to be related to functional roles. In Arabidopsis, FLAs containing a single FAS domain were found to be important for the integrity and elasticity of the plant cell wall matrix, and FLAs with two FAS domains and two AGP domains were found to be involved in maintaining proper cell expansion under salt stress conditions. The main purpose of the present work was to elucidate the expression pattern of selected FLA genes during embryo and seed development using RT-qPCR. AtFLA8 and AtFLA10, two Arabidopsis genes that stood out in previous microarray studies of embryo development, were further examined using promoter-driven gene reporter analyses. We also studied the expression of cork oak FLA genes and found that their expression partially parallels the expression patterns of the putative AtFLA orthologs. We propose that the functional organization of FLAs is conserved and may be related to fundamental aspects of embryogenesis and seed development across angiosperms. Phylogenetic studies were performed, and we show that the same basic four-subgroup organization described for Arabidopsis FLA gene classification is valid for most Arabidopsis FLA orthologs of several plant species, namely poplar, corn and cork oak.

Editorial Expression of Concern: Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

Editorial Expression of Concern: Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

Degradation of plant arabinogalactan proteins by intestinal bacteria: characteristics and functions of the enzymes involved.

Arabinogalactan proteins (AGPs) are complex plant proteoglycans that function as dietary fiber utilized by human intestinal bacteria such as Bifidobacterium and Bacteroides species. However, the degradative mechanism is unknown because of the complexity of sugar chains of AGPs as well as variation among plant species and organs. Recently, AGP degradative enzymes have been characterized in Bifidobacterium and Bacteroides species. In this review, we summarize the characteristics and functions of AGP degradative enzymes in human intestinal bacteria.

Cell Wall Polymer Composition and Spatial Distribution in Ripe Banana and Mango Fruit: Implications for Cell Adhesion and Texture Perception.

Banana (Musa acuminata) and mango (Mangifera indica) are two of the most popular fruits eaten worldwide. They both soften during ripening but their textural attributes are markedly different. This study aimed to elucidate the molecular mechanism underpinning textural differences between banana and mango. We used a novel combination of methods at different scales to analyse the surface properties of fruit cells and the potential contribution of cells and cell wall components to oral processing and texture perception. The results indicated that cell separation occurred easily in both organs under mild mechanical stress. Banana cells showed distinctively elongated shapes with distinct distribution of pectin and hemicellulose epitopes at the cell surface. In contrast, mango had relatively spherical cells that ruptured during cell separation. Atomic force microscopy detected soft surfaces indicative of middle lamella remnants on banana cells, while mango cells had cleaner, smoother surfaces, suggesting absence of middle lamellae and more advanced cell wall disassembly. Comparison of solubilized polymers by cell wall glycome analysis showed abundance of mannan and feruylated xylan in separation exudate from banana but not mango, but comparable levels of pectin and arabinogalactan proteins. Bulk rheology experiments showed that both fruits had similar apparent viscosity and hence might be extrapolated to have similar “oral thickness” perception. On the other hand, oral tribology experiments showed significant differences in their frictional behavior at orally relevant speeds. The instrumental lubrication behavior can be interpreted as “smooth” mouthfeel for mango as compared to “astringent” or “dry” for banana in the later stages of oral processing. The results suggest that cell wall surface properties contribute to lubricating behavior associated with textural perception in the oral phase.

Arabinogalactan protein–rare earth element complexes activate plant endocytosis

Endocytosis is essential to all eukaryotes, but how cargoes are selected for internalization remains poorly characterized. Extracellular cargoes are thought to be selected by transmembrane receptors that bind intracellular adaptors proteins to initiate endocytosis. Here, we report a mechanism for clathrin-mediated endocytosis (CME) of extracellular lanthanum [La(III)] cargoes, which requires extracellular arabinogalactan proteins (AGPs) that are anchored on the outer face of the plasma membrane. AGPs were colocalized with La(III) on the cell surface and in La(III)-induced endocytic vesicles in Arabidopsis leaf cells. Superresolution imaging showed that La(III) triggered AGP movement across the plasma membrane. AGPs were then colocalized and physically associated with the μ subunit of the intracellular adaptor protein 2 (AP2) complexes. The AGP-AP2 interaction was independent of CME, whereas AGP's internalization required CME and AP2. Moreover, we show that AGP-dependent endocytosis in the presence of La(III) also occurred in human cells. These findings indicate that extracellular AGPs act as conserved CME cargo receptors, thus challenging the current paradigm about endocytosis of extracellular cargoes.

The colloidal stabilization of young red wine by Acacia senegal gum: The involvement of the protein backbone from the protein-rich arabinogalactan-proteins

The arabinogalactan-proteins (AGPs) from Acacia senegal gum are used as protective colloid in young red wines to prevent the precipitation of the coloring matter. Usually, Acacia senegal gum is chosen based on its efficiency to stabilize iron hexacyanoferrate salts in hydro-alcoholic solution. In this study, the protective activity of Acacia senegal gum and its macromolecular fractions (HIC–F1, HIC–F2 and HIC–F3), separated according to their hydrophobicity, towards the precipitation of iron hexacyanoferrate salts and polyphenols in hydro-alcoholic solutions are reported. AGPs prevent the colloidal instability of both iron hexacyanoferrate salts and polyphenols in “model” hydro-alcoholic solutions and young red wine with a good correlation between results obtained on both systems. This result strengthens the use of iron hexacyanoferrate salts (hydro-alcoholic – mineral test) for the evaluation of the protective activity of A. senegal gum in young red wine. The precipitation of iron hexacyanoferrate salts is avoided by the electrostatic binding of Ca2+, the driver of the instability, with the negatively charged carried by amino-acids from the protein backbone or both by amino acids and uronic acid monosaccharides localized close to the protein backbone of AGPs. The protective activity closely depends on the protein content of AGPs in both iron hexacyanoferrate salts and polyphenols hydro-alcoholic solutions: the more they are rich in proteins, the more their colloidal stabilizing efficiency is (HIC–F3>HIC–F2>HIC–F1). The differences observed in the protective activity between AGPs from the three HIC fractions are relied not only to their protein content but also to their related rate of glycosylation that modulates the protein accessibility to its environment, then their physicochemical properties as their hydrophobic behavior.

Tracking polysaccharides during white winemaking using glycan microarrays reveals glycoprotein-rich sediments.

Winemaking results in a significant amount of sediments that are formed in the tanks, the vats and in the bottles before and after fermentation. Little is known about the biochemical composition of these sediments apart from the fact that they are assumed to be derived in large part from the grape matrix. Glycan microarray technology offers a relatively rapid means to track the polysaccharides from their origin in the grape material and throughout the various steps in the winemaking process. In this study Comprehensive Microarray Polymer Profiling (CoMPP) was used to investigate the glycan-rich composition of particularly white grapes during winemaking and then investigate the effects of recombinant and commercial enzyme formulations on wine sediment compositions. The gross lees or sediments produced in the absence of enzymes were found to be composed of an abundance of homogalacturonans, rhamnogalacturonans, arabinans and galactans in addition to an abundance of extensins and arabinogalactan proteins. The addition of enzymes was shown to strip off the homogalacturonan and much of the rhamnogalacturonan with its side chains revealing a sediment layer composed almost exclusively of extensins and arabinogalactan proteins. The effect of winemaking techniques was shown to have an effect on the glycan-rich wine sediment compositions and holds implications for the management of gross lees in a winery environment.

Hydroxyproline-Rich Glycoproteins as Markers of Temperature Stress in the Leaves of Brachypodium distachyon.

Plants frequently encounter diverse abiotic stresses, one of which is environmental thermal stress. To cope with these stresses, plants have developed a range of mechanisms, including altering the cell wall architecture, which is facilitated by the arabinogalactan proteins (AGP) and extensins (EXT). In order to characterise the localisation of the epitopes of the AGP and EXT, which are induced by the stress connected with a low (4 °C) or a high (40 °C) temperature, in the leaves of Brachypodium distachyon, we performed immunohistochemical analyses using the antibodies that bind to selected AGP (JIM8, JIM13, JIM16, LM2 and MAC207), pectin/AGP (LM6) as well as EXT (JIM11, JIM12 and JIM20). The analyses of the epitopes of the AGP indicated their presence in the phloem and in the inner bundle sheath (JIM8, JIM13, JIM16 and LM2). The JIM16 epitope was less abundant in the leaves from the low or high temperature compared to the control leaves. The LM2 epitope was more abundant in the leaves that had been subjected to the high temperatures. In the case of JIM13 and MAC207, no changes were observed at the different temperatures. The epitopes of the EXT were primarily observed in the mesophyll and xylem cells of the major vascular bundle (JIM11, JIM12 and JIM20) and no correlation was observed between the presence of the epitopes and the temperature stress. We also analysed changes in the level of transcript accumulation of some of the genes encoding EXT, EXT-like receptor kinases and AGP in the response to the temperature stress. In both cases, although we observed the upregulation of the genes encoding AGP in stressed plants, the changes were more pronounced at the high temperature. Similar changes were observed in the expression profiles of the EXT and EXT-like receptor kinase genes. Our findings may be relevant for genetic engineering of plants with increased resistance to the temperature stress.

Adsorption of Hyperbranched Arabinogalactan-Proteins from Plant Exudate at the Solid–Liquid Interface

Adsorption of hyperbranched arabinogalactan-proteins (AGPs) from two plant exudates, A. senegal and A. seyal, was thoroughly studied at the solid–liquid interface using quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), and atomic force microscopy (AFM). Isotherms of the adsorption reveal that 3.3 fold more AGPs from A. seyal (500 ppm) are needed to cover the gold surface compared to A. senegal (150 ppm). The pH and salt concentration of the environment greatly affected the adsorption behavior of both gums, with the surface density ranging from 0.92 to 3.83 mg m−2 using SPR (i.e., “dry” mass) and from 1.16 to 19.07 mg m−2 using QCM-D (wet mass). Surprisingly, the mass adsorbed was the highest in conditions of strong electrostatic repulsions between the gold substrate and AGPs, i.e., pH 7.0, highlighting the contribution of other interactions involved in the adsorption process. Structural changes of AGPs induced by pH would result in swelling of the polysaccharide blocks and conformational changes of the polypeptide backbone, therefore increasing the protein accessibility and hydrophobic interactions and/or hydrogen bonds with the gold substrate.

Arabinose biosynthesis is critical for salt stress tolerance in Arabidopsis.

The capability to maintain cell wall integrity is critical for plants to adapt to unfavourable conditions. l-Arabinose (Ara) is a constituent of several cell wall polysaccharides and many cell wall-localised glycoproteins, but so far the contribution of Ara metabolism to abiotic stress tolerance is still poorly understood. Here, we report that mutations in the MUR4 (also known as HSR8) gene, which is required for the biosynthesis of UDP-Arap in Arabidopsis, led to reduced root elongation under high concentrations of NaCl, KCl, NaNO3 , or KNO3 . The short root phenotype of the mur4/hsr8 mutants under high salinity is rescued by exogenous Ara or gum arabic, a commercial product of arabinogalactan proteins (AGPs) from Acacia senegal. Mutation of the MUR4 gene led to abnormal cell-cell adhesion under salt stress. MUR4 forms either a homodimer or heterodimers with its isoforms. Analysis of the higher order mutants of MUR4 with its three paralogues, MURL, DUR, MEE25, reveals that the paralogues of MUR4 also contribute to the biosynthesis of UDP-Ara and are critical for root elongation. Taken together, our work revealed the importance of the Ara metabolism in salt stress tolerance and also provides new insights into the enzymes involved in the UDP-Ara biosynthesis in plants.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.