Jean-Claude Mollet
email: jean-claude.mollet@univ-rouen.fr

Flavien Dardelle

Arnaud Lehner

Yasmina Ramdani

Muriel Bardor

Patrice Lerouge

Azeddine Driouich

Biochemical and immunocytological characterizations of Arabidopsis thaliana pollen tube cell wall 1 [W]

During plant sexual reproduction, pollen germination and tube growth require a development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis thaliana pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactanprotein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-L-arabinan, the side chains of rhamnogalacturonan I.

Finally, MALDI-TOF MS analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono Oacetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that Arabidopsis pollen tube wall has its own characteristics compared to other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.

INTRODUCTION

Fertilization of flowering plants requires the delivery of the two sperm cells, carried by a fast growing tip polarized pollen tube, to the egg cell. In plants with dry stigma and solid style such as Arabidopsis thaliana, this process begins with the deposition and specific adhesion of the pollen grains on the stigmatic tissue, subsequent hydration of the pollen grains and germination of pollen tubes [START_REF] Palanivelu | Pollen tube targeting and axon guidance: parallels in tip growth mechanisms[END_REF]. Pollen tubes invade the papillae cell wall of the stigma, enter the short style, and grow through the apoplast of the specialized transmitting tract (TT) cells that is filled with a nutrient-rich extracellular matrix [START_REF] Kandasamy | Pollen-pistil interactions and developmental regulation of pollen tube growth in Arabidopsis[END_REF][START_REF] Lennon | The structure of the transmitting tissue of Arabidopsis thaliana (L.) and the path of pollen tube growth[END_REF]. During this invasive growth, pollen tubes are guided to the ovules via signals that need to pass through the cell wall to reach their membrane-associated or intracelllular targets [START_REF] Lord | The mechanisms of pollination and fertilization in plants[END_REF][START_REF] Kim | Chemocyanin, a small basic protein from the lily stigma, induces pollen tube chemotropism[END_REF][START_REF] Mccormick | Is there more than one way to attract a pollen tube?[END_REF][START_REF] Boavida | Gametophyte interaction and sexual reproduction: how plants make a zygote[END_REF][START_REF] Johnson | Extracellular guidance cues and intracellular signaling pathways that direct pollen tube growth[END_REF]. In plant species with wet stigma and hollow style such as lily, adhesion between the pollen tube wall and the TT epidermis extracellular matrix is important for the growth of the pollen tubes toward the ovules [START_REF] Mollet | A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix[END_REF][START_REF] Park | A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix[END_REF][START_REF] Chae | A relationship between structural features and lily pollen tube adhesion activity of SCA (Stigma/style Cysteine-rich Adhesin) isoforms[END_REF][START_REF] Mollet | Cell adhesion, separation and guidance in compatible plant reproduction[END_REF]. In addition of being the interface between the tube cells and the surroundings (female sporophyte or culture medium), the pollen tube wall also controls the cell shape, protects the generative cells and allows resistance against turgor pressure [START_REF] Geitmann | The Architecture and properties of the pollen tube cell wall[END_REF][START_REF] Geitmann | How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry[END_REF].

Most of our knowledge on cell wall polymers of higher plants comes from investigations on vegetative organs in which cells have diffuse growth. The cell wall is mainly composed of polysaccharides (cellulose, hemicellulose, pectin and occasionally callose, depending on the tissue) and proteoglycans (e.g. extensin and arabinogalactan proteins) forming a complex network with processing enzymes.

Pectins are complex wall macromolecules with uncertain supramolecular organization [START_REF] Vincken | If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture[END_REF] consisting of homogalacturonan (HG) that can be methyl-and acetylesterified, rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan [START_REF] Carpita | The cell wall[END_REF]. HG is a polymer of repeated units of (1→4)α-D-galacturonic acid that can be cross-linked with calcium upon block wise action of pectin methylesterases on methylesterified HG [START_REF] Micheli | Pectin methylesterases: cell wall enzymes with important roles in plant physiology[END_REF]. RG-II has the same homopolymer backbone as HG but is substituted with four different oligosaccharides composed of unusual sugars such as apiose, aceric acid, 3-deoxy-D-manno-2-octulosonic acid (Kdo) of unknown function (reviewed by [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF]. RG-I consists of the repeating disaccharide, (1→4)-α-D-galacturonic acid-(1→2)-α-L-rhamnose, unit with a wide variety of side chains attached to the rhamnosyl residues, ranging from monomers to large oligosaccharides such as (1→4)-β-D-galactan, (1→5)-α-L-arabinan, and/or type I arabinogalactan [START_REF] Caffall | The structure, function, and biosynthesis of plant cell wall pectic polysaccharides[END_REF].

Xyloglucan (XyG) is the major hemicellulosic polysaccharide of the primary wall of flowering plants. Classic XyG consists of a (1→4)-β-D-glucan backbone substituted with xylose, galactose-xylose or fucose-galactose-xylose motifs which correspond, according to the one letter code proposed by [START_REF] Fry | An unambiguous nomenclature for xyloglucan-derived oligosaccharides[END_REF], to X, L and F, respectively, G being the unsubtituted glucosyl residue of the glucan backbone. The main XyG fragments released after endo-glucanase treatment of the cell wall from wild type Arabidopsis vegetative organs are generally XXXG, XXLG/XLXG, XXFG and XLFG [START_REF] Zablackis | Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves[END_REF][START_REF] Lerouxel | Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting[END_REF][START_REF] Nguema-Ona | The reb1-1 mutation of Arabidopsis. Effect on the structure and localization of galactose-containing cell wall polysaccharides[END_REF][START_REF] Obel | Microanalysis of plant cell wall polysaccharides[END_REF]. In addition, O-acetylation of XyG can occur, most generally on the galactosyl residues, but its biological function is unknown [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]. In the primary wall, XyG interacts with cellulose microfibrils via hydrogen bonds and participate in the control of cell expansion [START_REF] Cosgrove | Enzymes and other agents that enhance cell wall extensibility[END_REF].

Arabinogalactan-proteins (AGPs) and extensin belong to the hydroxyprolin-rich glycoproteins (HRGPs) superfamily with very high level of type II arabinogalactan glycosylation [START_REF] Nothnagel | Proteoglycans and related components in plant cells[END_REF][START_REF] Showalter | Arabinogalactan-proteins: structure, expression and function[END_REF]. These proteoglycans have been implicated in many aspects of plant development including cell expansion, cell signaling and communication, embryogenesis, wound response and pollen tube guidance [START_REF] Wu | A floral transmitting tissue specific glycoprotein attracts pollen tubes and stimulates their growth[END_REF][START_REF] Nothnagel | Proteoglycans and related components in plant cells[END_REF][START_REF] Seifert | The biology of arabinogalactan proteins[END_REF][START_REF] Driouich | Intercourse between cell wall and cytoplasm exemplified by arabinogalactan proteins and cortical microtubules[END_REF].

Despite the importance of pollen tubes for the delivery of the sperm cells to the egg, little is known about the underlying molecular mechanisms that regulate the mechanical interaction of pollen tubes with female floral tissues. There are very scarce data concerning the different components of the pollen tube cell wall. Past approaches to characterize the pollen tube cell wall are limited to few plant species including Camellia [START_REF] Nakamura | Sugar composition of pollen grain and pollen tube cell walls[END_REF], Lilium [START_REF] Jauh | Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination[END_REF][START_REF] Mollet | Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside[END_REF], Nicotiana [START_REF] Rae | Composition of the cell walls of Nicotiana Mata Link et Otto pollen tubes[END_REF][START_REF] Li | Immunogold localization of arabinogalactan proteins, unesterified and esterified pectins in pollen grains and pollen tubes of Nicotiana tabacum L[END_REF][START_REF] Ferguson | Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum[END_REF][START_REF] Qin | Localization of arabinogalactan proteins in anther, pollen, and pollen tube of Nicotiana tabacum L[END_REF], Pinus [START_REF] Derksen | The wall of Pinus sylvestris L. pollen tubes[END_REF] and Zea [START_REF] Rubinstein | Extensin-like glycoproteins in the maize pollen tube wall[END_REF] and are mostly based on immunocytochemistry. These studies revealed that, depending on the species, the pollen tube cell wall contains epitopes that are found in the polymers described above including HGs with varying levels of methylesterification, AGPs, extensin-like proteins, and low amounts of cellulose. Unlike most other plant cells, callose, a (1→3)-β-glucan, is predominant and it is deposited in the wall back from the tip. Moreover, it is deposited in regular intervals to form callose plugs that maintain the tube cell in the apical expanding region of the tube and separate the viable from the degenerating region of the tube (reviewed by [START_REF] Geitmann | The Architecture and properties of the pollen tube cell wall[END_REF]. Only few reports have investigated the pollen tube of the model plant, Arabidopsis thaliana. They have focused either on in vivo or on in vitro grown pollen tubes using monoclonal antibodies (MAbs) directed against a subset of cell wall epitopes present in HG, XyG and AGPs [START_REF] Lennon | The in vivo pollen tube cell of Arabidopsis thaliana. I. Tube cell cytoplasm and wall[END_REF][START_REF] Freshour | Distribution of fucose-containing xyloglucans in cell walls of the mur1 mutant of Arabidopsis[END_REF][START_REF] Pereira | Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana[END_REF] but quantitative chemical analyses are lacking. This lack of information is most likely due to the fact that substantial amounts of pollen tube material is needed for chemical analysis and a reproducible and efficient method for liquid culture of Arabidopsis pollen tubes had not been established until recently [START_REF] Boavida | Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana[END_REF][START_REF] Daher | Optimization of conditions for germination of cold-stored Arabidopsis thaliana pollen[END_REF].

Here, we report the composition and localization of different cell wall polymers of in vitro-grown wild-type Arabidopsis pollen tubes based on biochemical analyses coupled to immunocytochemical investigations both at light and transmission electron microscope (TEM) levels using recently developed MAbs. Our results show distinct patterns of labeling (tip, whole tube, and shank of the tube) depending on the recognized epitope. The most striking observations are (1) the abundance of (1→5)-α-L-arabinan in the tube wall (> 40 mol% of arabinose), mostly localized, with LM6 and LM13, in the outer wall layer of the tube and (2) an atypical XyG MALDI-TOF MS profile with over 68% of the oligosaccharide fragments being O-acetylated.

RESULTS

In vitro pollen tube growth and β-glucan localization

In order to study the cell wall of Arabidopsis pollen tubes, sufficient material was necessary to perform the biochemical analysis. To achieve this, a large number of flowers, a good in vitro pollen germination rate with sufficient pollen tube length were required. As shown in Fig. 1A, high rates of pollen germination were obtained (67% ± 12) with an average of pollen tube length of 1248 µm ± 374 and a growth rate of 0.46 µm.min -1 ± 0.05 after 16h of culture in liquid medium. Moreover, 16h-old pollen tubes displayed typical tip organization (Fig. 1A insert) without any plasmolysis event. These results showed that using these in vitro conditions, pollen tube length is approaching what we should expect in in vivo condition given the distance between stigma and ovules. By comparison, 6h-old pollen tubes were fourfold shorter with an average length of 307 µm ± 102 and an estimated growth rate of 1.6 µm.min -1 ± 0.17. Cytochemical staining of β-glucans with aniline blue (for callose) and calcofluor white (mainly callose and cellulose) was performed in order to visualize the distribution of these βglucans along the tube wall and within the tube. Aniline blue staining was not detectable in the tip region and was mainly localized in the wall, back from the tip, and within the tube as callose plugs that are periodically synthesized (Fig. 1B; Table I). In contrast, staining with calcofluor white was uniform along the whole tube and the tip. The bright regions within the tubes revealed several plugs (Fig. 1C). At the ultrastructural level, the Arabidopsis pollen tube wall back from the tip appeared as a bi-layered structure consisting of a fibrillar outer and a weakly electron-dense inner wall layer (Fig. 2A). Immunogold localization of callose resulted in dense labeling in the inner wall layer back from the tip (Supplemental data, Fig. S1A).

Immunolocalization of Arabidopsis pollen tube cell wall epitopes

Immunolabeling pattern of pectin domains (Table I). Probing with JIM7 and JIM5 that recognize partially and highly methylesterified HGs revealed that JIM7 epitopes were dominantly localized in the tip region of the pollen tubes (Fig. 1D). Similar observations were made at tube tips emerging from pollen grains (Fig. 1D insert). In contrast, JIM5 staining was observed mostly back from the tip with brighter ring-like deposits presumably originating from successive temporary slow-downs in the growth of the tubes (Fig. 1E). LM8, specific for xylogalacturonan epitopes, uniformly labeled the whole pollen tube wall (Figs. 1F, 1F insert).

(1→5)-α-L-arabinan epitopes, localized with LM6 and LM13, were clearly detected in the entire pollen tube wall (Figs. 1G,1I) with stronger fluorescent signal at the pollen tube tip (Figs. 1H,1J). A ring-like structure was also noticeable as a collar at the emergence of the pollen tube from the pollen grain (Fig. 1G). Immunogold labeling of epitopes recognized by LM6 was mostly found in the fibrillar outer wall layer (Figs. 2B-2C). A strong concentration of gold particles was also observed over densely stained vesicles possibly associated with the trans Golgi network (Fig. 2B). Epitopes recognized by LM6 were also detected in the intine wall of the pollen grain (Supplemental data, Fig. S1B).

Immunolabeling of xyloglucan motifs.

To investigate the distribution of XyG, pollen tubes were probed with the MAbs LM15 and CCRC-M1 that recognize non-galactosylated (XXXG motif) and fucosylated XyG domains, respectively. As shown in Figs. 1K and1L, both MAbs bound to the entire pollen tube wall.

TEM observations showed that fucosylated XyG epitopes probed with the MAb CCRC-M1 were mainly found in the inner pollen tube wall (Fig. 2D), whereas the nongalactosylated XyG epitopes tagged with LM15 were detected in both the inner and outer layers of the wall (Figs. 2E-2F; Table I). Immunolabeling of pollen grains with the LM15 and CCRC-M1 MAbs resulted in a strong detection of XyG epitopes, mostly in the intine wall and at the vicinity of the plasma membrane (Supplemental data, Figs. S1C-S1D).

Immunolabeling of arabinogalactan-proteins. Epitopes of AGPs recognized by the MAb LM2 were found slightly labeled throughout the pollen tube wall with a stronger signal in the tip region (Figs. 1M-1N; Table I). Similar results were observed with MAC207 whereas JIM13 did not label the tube cell wall (Table I).

Immunolabeling at fluorescent microscopy level was carried out on 6h and 16h-old pollen tubes and did not show any difference in labeling pattern between the two time points (data not shown). Controls for immunofluorescent labeling did not show any fluorescence of the pollen tubes and a weak autofluorescence of the pollen grain exine (data not shown).

Controls for immunogold detection did not exhibit any significant non specific gold particles on the resin, pollen tubes and pollen grains (Supplemental data, Figs. S1E-S1H). A summary of immunolocalization data on the Arabidopsis pollen tube wall is shown in Table I.

Contribution of pollen grain wall versus pollen tube wall for biochemical analysis

We estimated the relative abundance of the pollen tube cell wall versus the pollen grain cell wall by measuring the cell wall thickness based on MET observations, pollen germination rates and pollen tube length (Supporting Table SI). The estimated ratio of the pollen grain cell wall on the pollen tube cell wall was about 1:7.4. Similarly, we also quantified by gas chromatography, the amount of carbohydrates present in non-germinated pollen grains and 16h-old germinated pollen tubes isolated from 520 flowers (Supplemental data, Fig. S2). Monosaccharide composition of the pollen grain and the pollen tube cell walls was similar and the amount of carbohydrates contained in the pollen grain cell wall was eightfold lower than the amount of carbohydrates contained in the pollen tube cell wall. These data indicate that the quantity of pollen grain cell wall material would interfere only weakly in the cell wall analysis and were used as a baseline for the monosaccharide composition of Arabidopsis pollen tubes.

Monosaccharide composition of the Arabidopsis pollen tube wall

Monosaccharide analysis of Arabidopsis pollen tube cell wall (Table II) displayed a high level of glucose (19.8 mol%) originating probably mostly from callose and to a lesser extent from XyG polymers. The most abundant monosaccharide, arabinose (43.6 mol%), and galactose (8.4 mol%) may come from AGP glycans and/or the pectic RG-I domain. Indeed, both sugars composing the backbone of RG-I (i.e. rhamnose and galacturonic acid) were present in the extract in equimolar amounts (~5 mol%). The remaining galacturonic acid (~5 mol%) composes undoubtedly the pectic HG backbone. XyG polymers generally consist of Xyl and Fuc residues that are also found in this analysis although in low amounts (1.2 mol% Fuc and 6.2 mol% Xyl) suggesting that XyG is not a major cell wall component of pollen tube cell wall.

The sequential extraction of the Arabidopsis pollen tube cell wall with ammonium oxalate and KOH aimed at yielding pectin-and hemicellulose-enriched fractions. Both extracts were analyzed for monosaccharide composition by gas chromatography (GC) and linkage/substitution sites by GC-mass spectrometry (MS) (Table II). The oxalate extract showed similar levels of uronic acid content by colorimetric assay (21.5 mol%) and GC (24.9 mol%) analysis. This extract was enriched in galacturonic acid (21.5 mol%) indicating the presence of the pectin domains HG and highly branched RG-I, as shown by the detection of 4.2 mol% of rhamnose, 24.7 mol% of arabinose and 18.3 mol% of galactose. These data are supported by the immuno-dot assay (Fig. 3) showing a strong interaction of the oxalate extract with JIM5 for partially methylesterified HG and LM6 for (1→5)-α-L-arabinan. This labeling was consistent with the presence of 28.2% of 5-Araf (Table II). Labeling with LM5 was weak which suggests a low amount of (1→4)-β-D-galactan. This observation was confirmed by the detection of only 1.5% of 4-linked galactosyl residues (Table II). Instead, galactosyl residues detected in the monosaccharide analysis originate probably from type II arabinogalactans as shown by the detection of 3-Gal, 6-Gal and 3,6-Gal (Table II).

In the KOH extract, levels of HG (galacturonic acid) and RG-I backbone (rhamnose and galacturonic acid) decreased significantly compared to the oxalate extract but the remaining RG-I domains were probably highly substituted by arabinans and/or arabinogalactans, as both arabinosyl and galactosyl residues counted for 37 mol% of the total sugars (Table II). The dot-blot immuno-assay (Fig. 3) revealed a significant reduction of the signal in the KOH extract with JIM5 compared to the oxalate extract indicating that KOH treatment had substantially removed methylester groups from the partially methylesterifed HG leaving low amount of non-esterified HG. In contrast, labeling for (1→5)-α-L-arabinan with LM6 was still strong (Fig. 3) and was confirmed with the detection of 14.6% of 5-Araf (Table II). The different components of XyG (i.e. Glc, Xyl, Gal and Fuc) were also present in the KOH extract (Table II), with a large increase in the glucose level (40.1 mol%) compared to the oxalate extract. As revealed by methylation analysis, the latter originated from XyG (2.2% 4,6-Glc), starch and/or amorphous cellulose (15.4% 4-Glc) but dominantly from callose with the presence of 3-Glc (27.6%) and possibly 2,3-Glc (7%) which may suggest that callose can be substituted on the C2 of the glucosyl residues (Table II). The MAb LM15 reacted strongly with both oxalate and KOH extracts (Fig. 3) suggesting that XyG were present in both extracts.

Matrix-Assisted Laser Desorption/Ionisation (MALDI) Time Of Flight (TOF) Mass

Spectrometry (MS) analysis of pollen tube cell wall xyloglucan fragments

Analysis by MALDI-TOF MS of XyG fragments released after xyloglucan endoglucanase treatment of Arabidopsis pollen tube cell wall (Fig. 4A) showed the typical ions, XXXG, XLXG/XXLG and XXFG (14.7%, 5.7% and 8.4%, respectively), found in XyG (Table III). However, these oligosaccharides were not the main motifs. Instead, the two principal ions identified (XLXG/XXLG and XXFG) present a shift of m/z = 42, characteristic of an O-acetyl group, branching presumably the galactosyl residue. These two O-acetylated fragments count for over 68% of the total oligosaccharide released. Overall, galactosylated and fucosylated xyloglucan oligosaccharides showed a relative abundance of 85% and 52%, respectively. By comparison, the main XyG fragment released after endo-glucanase treatment of Arabidopsis leaf cell wall was XXXG (Supplemental data, Fig. S3) and the total level of Oacetylated fragments was 33%. Moreover, based on the signal intensity, the ratio of Fuc to Xyl can be estimated to 1:5.7 similar to what was found in the compositional analysis of the pollen tube wall extract (1:5.2) which suggests that other xylose-containing polymers such as xylan may not be present or in very low amount in the pollen tube cell wall. Finally, monosaccharide analysis of the endo-glucanase-resistant pollen tube cell wall residue (Fig. 4B) revealed, despite a clear decrease in the carbohydrate content of Xyl, Gal and Fuc compared to the untreated cell wall material, that all the XyGs have not been completely degraded by the enzyme, perhaps due to their tight association with other cell wall components.

DISCUSSION

Since the AGI (2000), increasing genomic, transcriptomic and proteomic data on Arabidopsis have become available (Becker et al., 2003;[START_REF] Honys | Transcriptome analysis of haploid male gametophyte development in Arabidopsis[END_REF][START_REF] Tung | Genome-wide identification of genes expressed in Arabidopsis pistils specifically along the path of pollen tube growth[END_REF][START_REF] Qin | Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil[END_REF][START_REF] Zou | Comparative proteomic analysis of Arabidopsis mature pollen and germinated pollen[END_REF]. The use of male and female gametophyte or sporophyte mutants has allowed major insight into pollen formation, pollen tube growth, guidance and signaling [START_REF] Rhee | Microspore separation in the quartet3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation[END_REF][START_REF] Dong | Plantacyanin plays a role in reproduction in Arabidopsis[END_REF][START_REF] Escobar-Restrepo | The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception[END_REF], but the biosynthesis, the remodeling and the overall role of the cell wall of the tip growing pollen tube cell is far from being fully understood. Using the in vitro pollen tube method developed by [START_REF] Boavida | Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana[END_REF], we obtained high levels of pollen germination ranging from 55% to 79% with pollen tubes that can expand over 1.5 mm after 16h of culture.

Based on this, we decided to investigate one of the main components, the cell wall, of this tipgrowing cell by means of immunolocalization at light and electron microscopy levels and analytical polysaccharide biochemistry. Our results showed that (1) the pectin wall is enriched in HG and (1→5)-α-L-arabinan, and (2) O-acetylation of XyG fragments released by endoglucanase is high compared to other sporophytic tissues.

Most of the precursor studies on pollen tube wall of the so-called "pollen tube model plants" (i.e. Nicotiana or Lilium) employed MAbs recognizing the HG pectin domains (JIM5 and JIM7) and a subset of carbohydrate epitopes of the cell surface AGPs (mainly JIM13, MAC207, LM2 and JIM8). To date, only scarce reports on pollen tube wall have been published using other well-defined MAbs such as the anti-XyG (CCRC-M1; [START_REF] Freshour | Distribution of fucose-containing xyloglucans in cell walls of the mur1 mutant of Arabidopsis[END_REF] and, to our knowledge, none with the more recently produced MAbs on pollen tube wall.

Arabidopsis pollen tube wall is composed of an inner layer enriched in callose and an outer fibrillar layer

TEM observations of in vitro rapid frozen Arabidopsis pollen tubes showed the typical two wall layers at the shank of the tip (a fibrillar outer and a weakly electron-dense inner wall layer) as observed in in vitro-grown tobacco pollen tube [START_REF] Li | Immunogold localization of arabinogalactan proteins, unesterified and esterified pectins in pollen grains and pollen tubes of Nicotiana tabacum L[END_REF][START_REF] Ferguson | Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum[END_REF] and in vivo-grown pollen tube from lily [START_REF] Roy | High-pressure freezing improves the ultrastructural preservation of in vivo grown lily pollen tubes[END_REF] and Arabidopsis [START_REF] Lennon | The in vivo pollen tube cell of Arabidopsis thaliana. I. Tube cell cytoplasm and wall[END_REF][START_REF] Derksen | Growth and cellular organization of Arabidopsis pollen tubes in vitro[END_REF]. Our data and those of others have shown that callose is the main component of the inner wall layer but it is not detectable at the tip whereas cellulose is weakly detected [START_REF] Ferguson | Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum[END_REF].

Arabidopsis pollen tube wall contains a subset of AGPs recognized by LM2

We found that AGPs were labeled in the whole tube with a stronger signal at the tip with LM2 whereas JIM13 labeling was almost absent. In contrast, JIM13 labeled lily pollen tube at the tip [START_REF] Mollet | Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside[END_REF], uniformly or in a ring-like deposition along the tube of tobacco [START_REF] Li | Periodic deposition of arabinogalactan epitopes in the cell wall of pollen tubes of Nicotiana tabacum L[END_REF][START_REF] Qin | Localization of arabinogalactan proteins in anther, pollen, and pollen tube of Nicotiana tabacum L[END_REF]. [START_REF] Pereira | Expression of arabinogalactan protein genes in pollen tubes of Arabidopsis thaliana[END_REF] also noted a lack of labeling of Arabidopsis pollen tube with JIM13 unlike MAC207, which labels uniformly the whole tube [START_REF] Coimbra | Arabinogalactan proteins (AGPs) related to pollen tube guidance into the embryo sac in Arabidopsis[END_REF]. These divergent data depend on the MAb used (despite the apparent identical epitope, β-D-GlcpA-(1→3)-α-D-GalpA-(1→2)-L-Rha, recognized by MAC207 and JIM13) and the species investigated. These differences may suggest that the carbohydrate moieties of AGPs are species and/or cell type specific (vegetative and sperm cells) or that the cell wall organization is different in these species which may alter the accessibility of the epitopes.

HG pectin domains are present in different location in Arabidopsis pollen tube wall

Immunofluorescent labeling with JIM5 and JIM7 shows a pattern similar to that observed in pollen tubes from species possessing a solid style such as the Solanaceae (potato, tobacco, petunia), Oleaceae (jasmine) and Poaceae (corn) [START_REF] Li | Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants[END_REF][START_REF] Qin | Localization of arabinogalactan proteins in anther, pollen, and pollen tube of Nicotiana tabacum L[END_REF]. A dominant localization of highly methylesterified HG, recognized by JIM7, was present at the tip and a periodic patterning of low methylesterified HG epitopes, labeled with JIM5, was visible behind the tip region. Pollen tube wall contains 10 mol% of GalA, half of it being presumably dedicated to HG building, which explains the weak labeling by JIM5 along the wall compared to other species [START_REF] Jauh | Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination[END_REF][START_REF] Qin | Localization of arabinogalactan proteins in anther, pollen, and pollen tube of Nicotiana tabacum L[END_REF] even though reduced accessibility of the MAb to the epitopes may occur due to other polymers. In fact, the pectinenriched extract isolated from the pollen tube wall showed a strong interaction with JIM5 (Fig. 3). Highly methylesterified HG and other pectic motifs are deposited at the growing tip by Golgi-derived vesicles and de-esterified via pectin methylesterases (PMEs) in the wall back from the tip, to allow calcium cross linking of the carboxyl groups [START_REF] Geitmann | The Architecture and properties of the pollen tube cell wall[END_REF]. In the tobacco pollen tube, two PME isoforms have been localized in the Golgi, secretory vesicles and at the outer surface of the plasma membrane where they may deesterify HG [START_REF] Li | Detection and localization of pectin methylesterase isoforms in pollen tubes of Nicotiana tabacum L[END_REF]. Two Arabidopsis mutants, vanguard1 [START_REF] Jiang | VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract[END_REF] and Atppme1 [START_REF] Tian | Pollen-specific pectin methylesterase involved in pollen tube growth[END_REF], defective in two different pollen specific PMEs, showed slight reduction (about 20%) of overall PME activity, reduced growth compared to wild type pollen tubes but different pollen tube phenotypes. Vanguard1 pollen tubes were unstable in vitro resulting in tip bursting and in vivo vanguard1 plants show reduced male fertility. In contrast, atppme1 pollen tubes showed in vitro branching pattern with numerous tips but did not show male sterility in vivo. These reports suggest that these mutant pollen tubes have modified cell wall mechanical properties, despite a lack of either immunolocalization or biochemical data showing a modification in the methylesterification level of the HG motif. It is noteworthy to point out, as suggested by [START_REF] Jiang | VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract[END_REF], that these PMEs may also have a role in modifying the female TT wall to facilitate pollen tube progression through the female tissue.

AtPPME1 and VANGUARD1 are among a group of 15 other pollen specific genes that are separated in two groups (I and II) and encode putative PMEs containing either a catalytic domain only (5 genes), or containing a catalytic domain and a putative PME inhibitor (PMEI) domain (10 genes) [START_REF] Pelloux | New insights into pectin methylesterase structure and function[END_REF][START_REF] Chen | Roles of pectin methylesterases in pollen tube growth[END_REF]. More studies on these important protein members are required to understand the fine-tuning of demethylesterification of HG, to determine the interaction between PME and PMEI and their roles in modifying the pollen tube mechanics and sensing [START_REF] Bosch | Pectin methylesterase, a regulator of pollen tube growth[END_REF]. Low esterified HG have been implicated in important physiological processes such as cell attachment in vegetative cells or organs [START_REF] Bouton | QUASIMODO1 encodes a putative membrane-bound glycosyltranferase required for normal pectin synthesis and cell adhesion in Arabidopsis[END_REF][START_REF] Leboeuf | Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant quasimodo 1 suspension-cultured cells: implications for cell adhesion[END_REF][START_REF] Durand | The organization pattern of root border-like cells of Arabidopsis thaliana is dependent on cell wall homogalacturonan[END_REF] and, associated with a stigma/style cysteine-rich adhesin (SCA), a secreted plant lipid transfer protein (LTP), in lily pollen tube adhesion [START_REF] Mollet | A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix[END_REF][START_REF] Park | A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix[END_REF]. Several LTPs are present in the TT of the Arabidopsis pistil along the pollen tube path [START_REF] Tung | Genome-wide identification of genes expressed in Arabidopsis pistils specifically along the path of pollen tube growth[END_REF]. Recently, the LTP5, produced in Arabidopsis pollen tube and in the pistil TT, has been proposed to play important roles in maintaining cell polarity at the tube tip and adhesion-mediated guidance perhaps by interaction with pectins [START_REF] Chae | A gain-of-function mutation of Arabidopsis Lipid Transfer Protein 5 disturbs pollen tube tip growth and fertilization[END_REF].

Arabidposis pollen tube wall is enriched in pectin Arabinan

In addition to HG motifs, Arabidopsis pollen tube wall contains highly branched RG-I with arabinan. Our results based on immunolocalization using LM6 and LM13, both recognizing (1→5)-α-L-arabinan epitopes, and biochemical analysis indicate firstly, a strong and evenly distributed labeling in the shank of the tube with a slight increase of the signal in the tip region. Secondly, the most striking point is the high level of arabinosyl residues present in the pollen tube wall, oxalate and KOH extracts, which counts for over 40 mol% of the total analyzed sugar. Chemical analysis of the pollen tube cell wall from tobacco [START_REF] Rae | Composition of the cell walls of Nicotiana Mata Link et Otto pollen tubes[END_REF], revealed also a high level of arabinose (between 15.4 and 26.4 mol% depending on the hydrolysis method) mostly 5-linked and to a lesser extent 5,2-linked. Moreover, tobacco pollen tube wall displayed low level of galactosyl residues and uronic acids [START_REF] Rae | Composition of the cell walls of Nicotiana Mata Link et Otto pollen tubes[END_REF].

Similarly, Lilium, Camellia and Tulipa pollen tube wall showed also high levels of arabinosyl residues [START_REF] Nakamura | Sugar composition of pollen grain and pollen tube cell walls[END_REF]. Analysis of Golgi vesicles from Camellia pollen tubes were also enriched in arabinose, galactose and uronic acid [START_REF] Hasegawa | Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen of Camellia japonica[END_REF]. All together, our data suggest that Arabidopsis pollen tube wall contains short RG-I backbone harboring long chains of (1→5)-α-L-arabinans in the outer wall layer. The significance of this common feature (high level of arabinan) between the pollen tubes originating from nonrelated species is not known but may suggest an important role of this polymer in pollen tube biology. The role of arabinan side chains of RG-I is not clear. Indeed, mutation in ARABINAN DEFICIENT 1 gene (ARAD1), a putative arabinosyltransferase involved in the biosynthesis of pectic arabinan in Arabidopsis, showed high reduction of arabinan side chains of RG-I without any compensation by other polymers. However, no obvious phenotype was detected, suggesting that a loss of arabinan in RG-I does not affect vegetative growth [START_REF] Harholt | ARABINAN DEFICIENT 1 is a putative arabinosyltransferase involved in biosynthesis of pectic arabinan in Arabidopsis[END_REF]. On the other hand, plants exhibiting mutations in two Reversibly Glycosylated Peptides (RGP1 and RGP2) that may be involved in cell wall biosynthesis showed a strong defect in the inner pollen wall and pollen lethality [START_REF] Drakakaki | Arabidopsis Reversibly Glycosylated Polypeptides 1 and 2 are essential for pollen development[END_REF]. Interestingly, rice RGPs show strong amino acid sequence identity (~80%) with UDP-arabinopyranose mutase implicated in UDP-Arap and UDP-Araf interconversion [START_REF] Konishi | A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose[END_REF]. We might speculate that RGP may be part of the arabinan biosynthesis network but further biochemical studies are required to validate this as many other molecules contain arabinosyl residues such as arabinoxylans and arabinogalactans. In Commelina communis, arabinan side chains of RG-I have been implicated in guard cell opening and closing. The authors suggested that they may prevent HG polymers from forming tight associations [START_REF] Jones | Cell wall arabinan is essential for guard cell function[END_REF]. A similar effect may occur between the pollen tube wall and the TT cell wall. Finally, pectic arabinans have also been implicated in cell attachment [START_REF] Iwai | Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia nonorganogenic callus with loosely attached constituent cells[END_REF][START_REF] Orfila | Altered middle lamella homogalacturonan and disrupted deposition of (1→5)-α-L-arabinan in the pericarp of Cnr, a ripening mutant of tomato[END_REF][START_REF] Leboeuf | Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths[END_REF][START_REF] Leboeuf | Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant quasimodo 1 suspension-cultured cells: implications for cell adhesion[END_REF]Peňa and Carpita, 2004). Adhesion of pollen tubes to the TT cells may be an important cue for guiding the tube cell within the female tissue as was shown in the hollow style plant, lily [START_REF] Mollet | Cell adhesion, separation and guidance in compatible plant reproduction[END_REF]. Investigation on mutant pollen defective in arabinosyltransferase will undoubtedly give insight into the function of arabinan side chains of RG-I in pollen/pollen tube biology.

Arabidopsis pollen tube wall endo-glucanase-sensitive XyG is highly acetylated

MAbs directed against two different XyG epitopes labeled the whole tube mostly in the outer wall layer and the in the tip. Oligosaccharide fragments containing these two epitopes (XXXG and XXFG) were also detected with MALDI-TOF MS after pollen tube cell wall digestion. Interestingly, XyG analysis from vegetative organs (leaf, stem or root), revealed consistently that the main fragments are XXXG and XXFG [START_REF] Zablackis | Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves[END_REF][START_REF] Pauly | Changes in the structure of xyloglucan during cell elongation[END_REF][START_REF] Lerouxel | Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting[END_REF]. Arabidopsis pollen tube XyG shows distinct mass spectra with two major fragments detected (XLXG/XXLG and XXFG) both being substituted by one acetyl group, presumably linked to the galactosyl residue. The signal intensity of these two fragments consistently accounted for over 68%. Recent investigations on XyG from leaf tissues isolated after laser microdissection has allowed a more detailed analysis showing subtle modification of XyG [START_REF] Obel | Microanalysis of plant cell wall polysaccharides[END_REF]. As an example, the level of acetylation of XLFG fragments in the mesophyll tissue was significantly higher than in the vascular tissue but the overall acetylation level in these tissues did not exceed 35-40% [START_REF] Obel | Microanalysis of plant cell wall polysaccharides[END_REF].

Together, this indicates that subtle changes in the acetylation level of XyG can occur but the function of these hydrophobic groups in cell wall biology is still unknown.

XyG are known to interact with cellulose microfibrils. Despite the low amount of cellulose in the pollen tube wall [START_REF] Doblin | Pollen tubes of Nicotiana alata express two genes from different β-Glucan synthase families[END_REF], cellulose is present at the tip and plays an important role in stabilizing the pollen tube tip wall. Cellulase treatment on growing pollen tubes resulted in tip swelling and eventually bursting (Aouar et al., 2010). Cellulose at the tip region may also serve as an interacting partner for XyG cross-linking. Acetylation of XyG does not seem to play a major role in the interaction with cellulose. In vitro experiments have shown that native (acetylated) and de-acetylated XyG were able to cross-link cellulose microfibrils in similar manner [START_REF] Whitney | Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose[END_REF]. In contrast, the high molecular weight of the XyG (880 kDa) and the presence of galactosyl residues appeared to be necessary to promote the interaction with cellulose. A lack of galactose in the XyG resulted in the selfassociation of XyG polymers instead of the interaction between XyG and cellulose [START_REF] Whitney | Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose[END_REF]. Similarly, Arabidopsis mutants with XyG lacking xylosyl, galactosyl or fucosyl residues showed reduction of tensile strength (Peňa et al., 2004) or abnormal bulging in the tip growing root hairs possibly due to impaired cellulose-XyG assembly [START_REF] Nguema-Ona | The reb1-1 mutation of Arabidopsis. Effect on the structure and localization of galactose-containing cell wall polysaccharides[END_REF][START_REF] Zabotina | Arabidopsis XXT5 gene encodes a putative α-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis[END_REF]. A similar phenotype was also noticed in plants exhibiting double mutations in the two XyG xylosyltransferases 1 and 2 genes (XXT1 and XXT2) that are apparently lacking XyG [START_REF] Cavalier | Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component[END_REF]. In the moss Physcomitrella, AGPs were found to be enriched in 3-O-Me-rhamnosyl residues compared to AGPs from more evolved plant species. This suggests a possible hydrophobic interaction of AGPs, which for a long time had been considered to have large capacity of interacting with water [START_REF] Fu | Physcomitrella patens arabinogalactan proteins contain abundant terminal 3-O-methyl-L-rhamnosyl residues not found in Angiosperms[END_REF]. All together, acetyl groups of XyG may promote hydrophobic interactions with other molecules within the pollen tube cell wall but also with other female components of the TT extracellular matrix during the intrusive growth of pollen tubes. Moreover, acetylation may modulate water uptake due to the non-polar property of these groups, but this requires further investigation.

In summary, this study provides a solid foundation for the use of Arabidopsis pollen tubes in cell wall biology and for the investigation of the male gametophyte cell elongation with mutants impaired in proteins implicated in the biosynthesis of specific cell wall polymers (XyG, HG, RG-I, RG-II and AGPs), cell wall deposition and cell wall remodeling. It may also help to dissect the mechanisms controlling the interaction between the pollen tube wall with the female counterpart. The challenge in this field is to overcome the generally observed phenotypes such as arrest of pollen formation, lack of pollen viability, absence of pollen tube germination or growth resulting in reduced male fertility when mutations are located in important male gametophyte genes [START_REF] Lalanne | SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in Arabidopsis[END_REF][START_REF] Iwai | The gene responsible for borate cross-linking of pectin rhamnogalacturonan-II is required for plant reproductive tissue development and fertilization[END_REF][START_REF] Drakakaki | Arabidopsis Reversibly Glycosylated Polypeptides 1 and 2 are essential for pollen development[END_REF][START_REF] Delmas | The synthesis of the rhamnogalacturonan II component 3-deoxy-D-manno-2octulosonic acid (Kdo) is required for pollen tube growth and elongation[END_REF][START_REF] Boavida | A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana[END_REF]. Chemical genetic screens on in vitro-grown pollen tubes may be an alternative. Nevertheless, studies on the two distinct features of Arabidopsis pollen tube wall (high levels of pectic arabinan side chains and XyG acetylation) require closer attention and investigation to better understand their possible roles in pollen tube growth, signaling, guidance and adhesion.

MATERIAL AND METHODS

Plant materials

Arabidopsis thaliana ecotype Columbia seeds stored at 4°C were spread on the surface of a sterile soil and cultured in a growth chamber with a photoperiod of 16h light/8h dark cycle at 20°C during the light phase and at 16°C in the dark phase with 60% humidity and daily watering. Only recently open flowers were collected.

In vitro pollen tube growth

Pollen was grown in vitro in a liquid medium according to the method described by [START_REF] Boavida | Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana[END_REF]. Briefly, flowers (forty per 1.5 mL tube) were submerged in 1 mL of germination medium (GM) containing 5 mM CaCl 2 , 2H 2 O, 0.01% (w/v) H 3 BO 3 , 5 mM KCl, 1 mM MgSO 4 , 7H 2 O and 10% (w/v) sucrose, pH 7.5. Tubes were shaken with a vortex to release the pollen grains from the anthers. Flowers were removed with a pair of tweezers and the pollen suspension was then pelleted down at 3200g for 6 min. New GM (250 µL) was added to the pellet and pollen grains were transferred into glass vials (14 x 45 mm) and grown in a growth chamber, in the dark at 22°C. For large scale culture, pollen from 200 flowers was grown in glass vials (25 x 50 mm) in 1.25 mL of GM. Using this harvesting method, the number of pollen grains collected per flower was estimated to 291 ± 63.3 (n = 10 independent experiments). Pollen tubes were grown for 6h for TEM sample preparation, 6h and 16h for immunolabeling at fluorescent microscopy level and 16h for biochemical analysis. Before any further manipulation, pollen germination and pollen tube growth were assessed under an inverted microscope.

Cytochemical staining

Calcofluor white (0.01% w/v) and decolorized aniline blue (0.1% w/v) in 100 mM K 3 PO 4 pH 11 [START_REF] Sa | A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically expressed genes[END_REF] were used to localize β-glucans (cellulose and callose) and callose, respectively.

Monoclonal antibodies

JIM13, MAC207 and LM2 MAbs recognize a carbohydrate moiety of AGPs [START_REF] Yates | Characterization of carbohydrate structural features recognized by anti-arabinogalactanprotein monoclonal antibodies[END_REF]. Extensins were probed with LM1 [START_REF] Smallwood | An epitope of rice threonine-and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins[END_REF]. The MAbs JIM5 and JIM7 recognize different levels of esterification of HG regions of pectins [START_REF] Clausen | Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7[END_REF]. (1→4)-β-D-galactans were probed with LM5 [START_REF] Jones | Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1-4)-beta-D-galactan[END_REF] and (1→5)-α-Larabinans with LM6 [START_REF] Willats | Generation of a monoclonal antibody specific to (1-5)-alpha-L-arabinan[END_REF] and LM13 [START_REF] Verhertbruggen | Developmental complexity of arabinan polysaccharides and their processing in plant cell walls[END_REF]. The MAbs directed against XyG were CCRC-M1 which recognizes an α-Fuc-(1→2)-β-Gal epitope [START_REF] Puhlmann | Generation of monoclonal antibodies against plant cell wall polysaccharides. I. Characterization of a monoclonal antibody to a terminal alpha-(1,2)-linked fucosyl-containing epitope[END_REF] and LM15 that binds to XXXG motif [START_REF] Marcus | Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls[END_REF]. MAbs were kindly provided by P. Knox (Univ of Leeds, UK) and M. Hahn (CCRC, Univ of Georgia, USA) or purchased at Plant Probes (Leeds, UK). Finally, callose was localized with a mouse anti-(1→3)-β-glucan (Biosupplies Australia).

Immunolocalization of Arabidopsis pollen tube wall epitopes

Pollen tubes in GM were mixed v/v with a fixation medium containing 100 mM PIPES buffer, pH 6.9, 4 mM MgSO 4 7H 2 O, 4 mM EGTA, 10% (w/v) sucrose, 5% (w/v) formaldehyde and incubated for 90 min at room temperature. Pollen tubes were rinsed 3 times by centrifugation with 50 mM PIPES buffer, pH 6.9, 2 mM MgSO 4 7H 2 O, 2 mM EGTA and 2.7 mM KCl, pH 7.4) supplemented or not with 3% fat free milk. Primary antibodies were diluted at 1/5 or 1/10 as previously described [START_REF] Mollet | Arabinogalactan proteins, pollen tube growth, and the reversible effects of Yariv phenylglycoside[END_REF] with PBS (with or without 3% milk). Pollen tubes were rinsed with the buffer and incubated overnight at 4°C in the dark with the secondary antibody combined with fluorescein isothiocyanate (FITC, Sigma) diluted at 1/50 with the appropriate buffer for 3h at 30°C. For JIM, LM and MAC antibody detection, goat anti-rat (immunoglobulin G, whole molecule)-FITC was used, for CCRC antibody detection, sheep anti-mouse (immunoglobulin G, whole molecule)-FITC was used. Controls were carried out by incubation of the pollen tubes with the secondary antibody only.

Microscope observation and acquisition of pollen tube pictures

Pollen tubes were observed under Nomarski differential interference contrast (DIC) optics or fluorescence illumination on a Leica DLMB microscope equipped with FITC (absorption, 485-520 nm; emission, 520-560 nm wavelength) or calcofluor white filter sets.

Pictures were acquired with a Leica DFC300FX camera.

Pollen count and germination rate were estimated with a Nageotte chamber. A pollen grain was considered germinated if the pollen tube length was greater than the pollen grain diameter. Pollen tube length was measured from the picture using the nih program ImageJ (Abramoff et al., 2004). At least 350 pollen tubes were measured in three independent experiments.

Electron microscopy preparation of in vitro grown pollen tubes

High pressure freezing/Freeze substitution (HPF/FS) sample preparation.

Centrifuged 6h-old pollen tubes were transferred into the cavity of gold cupules (200 μm in depth and 1.2 mm in diameter) coated with soybean lecithin (100 mg.mL -1 in chloroform).

Excess medium was removed using a filter paper. Then, samples were frozen using a highpressure freezing EM-PACT (Leica), according to a maximum cooling rate of 10000°C/s, incoming pressure of 7.5 bars and working pressure of 4.8 bars. Cupules containing frozen samples were stored in liquid nitrogen until the freeze substitution procedure was initiated.

After high-pressure freezing, samples were transferred to a freeze substitution automate (EM-AFS, Leica) precooled to -140°C. Freeze substitution conditions followed a modified procedure from D. Studer (personal communication). Substitution media were composed of 2% osmium in anhydrous acetone. Samples were substituted at -90°C for 72h.

The temperature was gradually raised (2°C.h -1 ) to -60°C and stabilized during 12h, then gradually raised (2°C.h -1 ) to -30°C (12h) and gradually raised again (2°C.h -1 ) to 0°C for 2h.

Samples were washed at room temperature with fresh anhydrous acetone. Infiltration was done at +4°C in acetone-Spurr resin (ratio 2:1; 1:1; 1:2, 8h each step) and with pure resin for at least 2 days. Polymerization was performed at 60°C for 16h. Using an ultracut EM-UC6 (Leica), thin sections (90 nm) were mounted on formvar-coated nickel grids.

Immunogold labeling and TEM observation. Grids were rehydrated in a TBS + BSA 0.2% buffer and blocked in a TBS/BSA 0.2%/milk 3% solution for 30 min. After 3 brief rinses in TBS/BSA 0.2% solution, grids were incubated 3h at 25°C in primary antibodies: non-diluted for LM15 and CCRC-M1 or diluted (LM6: 1/2; anti-callose: 1/100) in TBS/BSA 0.2% buffer. Then, grids were washed (6 x 5 min) in TBS/BSA 0.2% and incubated for 1h at 25°C in a 1/20 secondary antibody (goat anti-rat for LM6 and LM15 or goat anti-mouse for CCRC-M1 and anti-callose) conjugated to 10 nm gold particles (British Biocell Intl). Finally, grids were washed 6 x 5 min in a TBS + BSA 0.2% buffer, 1 min in TBS, 10 min in TBS + glutaraldehyde 2%, 5 min in TBS then 2 x 5 min in ddH 2 O.

The sections were stained with 0.5% (w/v) uranyl acetate in methanol for 10 min in the dark, rapidly rinsed 10 times with water and stained with lead citrate for 10 min and briefly rinsed 10 times with water.

Grids were observed at 80 kV with a TEM (Tecnai 12 Bio-Twin, Philips, FEI) and pictures were acquired with an Erlangshen ES500W camera (Gatan).

Cell wall extraction of Arabidopsis pollen tubes

16h-old pollen tubes from pollen grains from 8500 flowers collected manually were pooled after addition of three volumes of EtOH 95% to the GM. This experiment was performed three times to allow three independent replicates. Pollen tubes were centrifuged at 5000g and rinsed three times with EtOH 70% to remove salts and sucrose from the GM. The insoluble material (3 x 12 mg) was ground and then treated three times with EtOH 70% at 70°C for 15 min followed by incubation with a mixture of chloroform:methanol (1:1, v/v).

The remaining insoluble material was then dried to yield the cell wall fraction (3 x 10 mg). As baseline control, cell wall from non-germinated pollen grains was extracted. A similar extraction method was applied to Arabidopsis mature rosette leaves to yield a leaf wall extract except that an acetone incubation was added after the chloroform:methanol treatment for pigment removal. This extract was used to compare the XyG fragments analyzed by MALDI-TOF MS with the pollen tube cell wall extract.

As described by [START_REF] Ray | Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif[END_REF], a pectin enriched fraction was obtained by extraction with boiling ammonium oxalate 0.5% (w/v) for 1h. After centrifugation, the supernatant was dialyzed against ddH 2 O and freeze-dried. The ammonium oxalate insoluble residue was then treated with 4M KOH supplemented with 20 mM NaBH 4 at room temperature for 12h. The alkaline soluble fraction was acidified to pH 5.5 with acetic acid, dialyzed against water and freeze-dried, to yield a hemicellulose-enriched fraction.

Analysis of cell wall polysaccharides

Photometric assays. Total uronic acid and neutral sugars were estimated with the mhydroxydiphenyl assay [START_REF] Blumenkrantz | New method for quantitative determination of uronic acids[END_REF] and the phenol sulfuric method [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF] with galactose and galacturonic acid as standards. Estimation of uronic acid and neutral sugar contents were calculated after correction of the interference of galacturonic acid with the phenol sulfuric and galactose with the mHDP assays.

Immuno-dot blot assays. Ammonium oxalate and KOH extracts (8, 4, 1 and 0.5 µg) were blotted onto nitrocellulose membrane. Immuno-dot blots were processed according to [START_REF] Jauh | Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination[END_REF]. Arabinan from sugar beet (Megazyme), gum arabic from acacia (Fisher), pectin with 8.6% O-methylation from citrus fruits (Sigma) and XyG from tamarind seeds (Megazyme) were used as controls. MAb binding was revealed with a goat anti-rat IgG (whole molecule) antibody conjugated with alkaline phosphatase (Sigma) diluted 1:1000 and developed with the NBT-BCIP kit (Promega).

Gas chromatography mass spectrometry. Monosaccharide compositions of the total cell wall, pectin and hemicellulose enriched fractions were determined by gas liquid chromatography according to [START_REF] York | Isolation and characterization of plant cell walls and cell wall components[END_REF] using inositol as internal standard. Briefly, each fraction (500 µg) was treated with 1 M methanolic-HCl at 80°C for 16h and the free monosaccharides were converted to their methyl glycosides. After silylation at 110°C for 20 min, samples were dried, dissolved in cyclohexane, and analyzed using a GC 3800 Varian gas chromatography system equipped with a DB1 capillary column and a flame ionization detector. A temperature program optimized for separation of the most common cell wall monosaccharides (Ara, Fuc, Gal, GalA, Glc, GlcA, Man, Rha, Xyl) was used. Data were analyzed and integrated using Varian GC Star Workstation software with the quantity of each monosaccharide corrected according to its response factor.

Preparation and linkage analysis of the partially methylated alditol acetates of pectin-and hemicellulose-enriched fractions were performed according to [START_REF] Smith | Structural profiling of oligosaccharides of glycoproteins[END_REF] except that the ultrasonic bath was performed for 1h instead of 15 min, dichloromethane was used instead of chloroform and a stream of air was used to dry samples instead of nitrogen. The resulting partially methylated alditol acetates were separated by GC (Hewlett-Packard 6890) on a Optima 5-MS capillary column (length 30 m, i.d. 0.25 mm, Macherey Nagel) and analysed by electron impact mass spectrometry using an Autospec mass spectrometer (Micromass) equipped with a Opus 3.1 data system.

Preparation of XyG oligosaccharides. 0.5 mg of pollen tube cell wall or leaf cell wall extracts were incubated under agitation for 16h at 37°C with 500 µL of an endo-(1→4)β-D-glucanase (5 Units, EC 3.2.1.4, Megazyme) prepared in 10 mM ammonium acetate buffer pH 5.0. Glucanase-resistant material was removed by centrifugation after addition of EtOH to reach a final concentration of 80%. The EtOH soluble XyG oligosaccharides were concentrated by evaporation under a stream of air.

MALDI-TOF mass spectrometry analysis of xyloglucan derived oligosaccharides.

MALDI-TOF mass spectra were acquired on a Voyager DE-Pro MALDI-TOF instrument (Applied Biosystems) equipped with a 337 nm nitrogen laser. Mass spectra were performed in the reflector delayed extraction mode using 2,5-dihydroxybenzoic acid (Sigma) as matrix. The matrix, freshly dissolved at 5 mg.mL -1 in a 70:30 acetonitrile/ 0.1% TFA, was mixed with the water solubilized oligosaccharides in a ratio 1:1 (v/v). These spectra were recorded in a positive mode, using an acceleration voltage of 20,000 V with a delay time of 100 ns and above 50% of the laser energy. They were externally calibrated using commercially available mixtures of peptides and proteins (Applied Biosystems). In this study, the spectra were calibrated using des-Arg 1 -bradykinin (904.4681 Da), angiotensin I (1296.6853), Glu 1fibrinopeptide B (1570.6774 Da), ACTH18-39 (2465.1989 Da). Laser shots were accumulated for each spectrum in order to obtain an acceptable signal to noise ratio (sum of 10 spectra of 1000 shots per spectrum).

Supplemental data

Supplemental Table SI. Estimation of the amount of pollen grain wall versus pollen tube cell wall. to the total of the K + and Na + adduct fragments. Oxalate and KOH extracts (8, 4, 1 and 0.5 µg) were probed with JIM5 for partially methylesterified HG, LM5

for (1→4)-β-D-galactan, LM6 for (1→5)-α-L-arabinan and LM15 for non galactosylated XyG (XXXG).

Controls (8 µg): a, arabinan from sugar beet; b, pectin with 8.6% methylesterification from citrus; c, XyG from tamarind seed; d, gum arabic from acacia.

Figure 1 .

 1 Figure 1. In vitro pollen tube growth, β-glucan staining and immuno-localisation of Arabidopsis pollen tube wall epitopes. A, 16h-old in vitro pollen tubes grown in liquid medium (arrows show callose plugs). Insert is a close up of 16h-old in vitro pollen tube tip. B, Cytochemical staining of β-glucan (callose) with aniline blue showing a callose plug (arrow) and a lack of staining in the pollen tube tip region (arrowhead). C. Cytochemical staining with calcofluor white showing callose plugs (arrow) and the localization of β-glucans in the wall. D-N, Immuno-fluorescence labeling of cell wall polymer epitopes at the surface of pollen tubes. Arabidopsis pollen tube wall epitopes probed with anti-pectin MAbs JIM7 (D), JIM5 (E) and LM8 (F), specific for low and highly methylesterified HG and xylogalacturonan, respectively. D, JIM7 tag showed a strong signal at the tip in welldeveloped pollen tube and emerging tube tip from the pollen grain (insert). E, pollen tube stained with JIM5 showing a very weak intensity of labeling at the tip (arrowhead) compared to the wall back from the tip. F, LM8 labeled evenly the entire pollen tube wall. Insert is a close up of in vitro pollen tube tip. G-J, Detection of (1→5)-α-L-arabinan epitopes with LM6 (G, H) and LM13 (I, J). Labeling was evenly distributed along the entire tube wall with a strong signal at the pollen tube tip. Note a collar-like structure (*) labeled at the emergence of the pollen tube from the pollen grain in G. K-L, Immuno-fluorescence staining of the non galactosylated (XXXG motif) and fucosylated XyG with LM15 (K) and CCRC-M1 (L), respectively. Labeling with both MAbs was not evenly detected in the wall and displayed periodic deposition of the epitopes containing polysaccharides during pollen tube growth. M-N, LM2 which recognizes β-D-GlcpA-(1→3)-α-D-GalpA-(1→2)-L-Rha moieties of AGPs labeled weakly the whole tube with the strongest signal at the tip. In M, a more intense labeling (*) was observed at the emergence of the tube from the pollen grain. pg, pollen grain. Pollen tubes were grown for 6h except in A and C (16h). Scale bars = 50 µm (A-C) and 5 µm (A insert; D-N).

Figure 2 .

 2 Figure 2. Electron micrographs showing the ultrastructure (A) and immunogold labeling of cell wall epitopes (B-F) of high pressure frozen/freeze substituted Arabidopsis pollen tube grown in vitro for 6h. A, Cross section of pollen tube showing the cell wall (cw) composed of two distinct layers: a fibrillar outer wall (ow) and a weakly electron-dense inner wall (iw). Well preserved organelles are also clearly distinguishable including endoplasmic reticulum (er), Golgi stacks (g), mitochondria (m) and vacuoles (v). pm, plasma membrane. B-C, Immunogold labeling of (1→5)-α-L-arabinan epitopes with LM6. Gold particles (arrowheads) are mostly localized in the outer wall (ow) layer. In B, possible trans Golgi network (tgn) and secretory vesicles (sv) labeled with LM6. D, Immunogold labeling of fucosylated xyloglucan motif recognized by CCRC-M1 in the inner (iw) and outer wall (ow) layers. Note the presence of gold particles in vesicles in the vicinity of the plasma membrane (white arrow). E-F, Immunogold labeling of non fucosylated xyloglucan motif (XXXG) with LM15. Gold particles (arrowheads) are visible in the inner (iw) and mainly in the outer walls (ow). cw, cell wall; er, endoplasmic reticulum; g, Golgi stacks; iw, inner wall layer; m, mitochondria; ow, outer wall layer; pm, plasma membrane; sv, secretory vesicles; tgn, trans Golgi network; v, vacuoles. Scale bars = 1 µm (A), 0.5 µm (B, D, E) and 100 nm (C and F).

Figure 3 .

 3 Figure 3. Immuno-dot blot assay of pectin (oxalate extract) and hemicellulose (KOH extract) enriched fractions isolated from Arabidopsis pollen tube cell wall.

Figure 4 .

 4 Figure 4. Analysis of Arabidopsis pollen tube XyG. A. MALDI-TOF mass spectrum of endo-glucanasegenerated XyG fragments from the cell wall of 16h-grown Arabidopsis pollen tubes. The structures of the XyG fragments are shown according to the nomenclature proposed by Fry et al. (1993). Underlined and bold structures represent O-acetylated side chains (+ 1 OAc). *, indicates the signal of XLFG fragment with K + adduct ion instead of Na + . B. Monosaccharide composition of 16h-grown Arabidopsis pollen tube cell wall before (untreated) and after (treated) endo-glucanase treatment. Only the monosaccharides composing XyG are shown: Fuc, fucose; Gal, galactose; Glc, glucose; Xyl, xylose. Others include arabinose, galacturonic acid, glucuronic acid, mannose and rhamnose.

Figure 1 .

 1 Figure 1. In vitro pollen tube growth, β-glucan staining and immuno-localisation of Arabidopsis pollen tube wall epitopes. A, 16h-old in vitro pollen tubes grown in liquid medium (arrows show callose plugs). Insert is a close up of 16h-old in vitro pollen tube tip. B, Cytochemical staining of β-glucan (callose) with aniline blue showing a callose plug (arrow) and a lack of staining in the pollen tube tip region (arrowhead). C. Cytochemical staining with calcofluor white showing callose plugs (arrow) and the localization of β-glucans in the wall. D-N, Immuno-fluorescence labeling of cell wall polymer epitopes at the surface of pollen tubes. Arabidopsis pollen tube wall epitopes probed with anti-pectin MAbs JIM7 (D), JIM5 (E) and LM8 (F), specific for low and highly methylesterified HG and xylogalacturonan, respectively. D, JIM7 tag showed a strong signal at the tip in welldeveloped pollen tube and emerging tube tip from the pollen grain (insert). E, pollen tube stained with JIM5showing a very weak intensity of labeling at the tip (arrowhead) compared to the wall back from the tip. F, LM8 labeled evenly the entire pollen tube wall. Insert is a close up of in vitro pollen tube tip. G-J, Detection of (1→5)-α-L-arabinan epitopes with LM6 (G, H) and LM13 (I, J). Labeling was evenly distributed along the entire tube wall with a strong signal at the pollen tube tip. Note a collar-like structure (*) labeled at the emergence of the pollen tube from the pollen grain in G. K-L, Immuno-fluorescence staining of the non galactosylated (XXXG motif) and fucosylated XyG with LM15 (K) and CCRC-M1 (L), respectively. Labeling with both MAbs was not evenly detected in the wall and displayed periodic deposition of the epitopes containing polysaccharides during pollen tube growth. M-N, LM2 which recognizes β-D-GlcpA-(1→3)-α-D-GalpA-(1→2)-L-Rha moieties of AGPs labeled weakly the whole tube with the strongest signal at the tip. In M, a more intense labeling (*) was observed at the emergence of the tube from the pollen grain. pg, pollen grain. Pollen tubes were grown for 6h except in A and C (16h). Scale bars = 50 µm (A-C) and 5 µm (A insert; D-N).

Figure 2 .

 2 Figure 2. Electron micrographs showing the ultrastructure (A) and immunogold labeling of cell wall epitopes (B-F) of high pressure frozen/freeze substituted Arabidopsis pollen tube grown in vitro for 6h. A, Cross section of pollen tube showing the cell wall (cw) composed of two distinct layers: a fibrillar outer wall (ow) and a weakly electron-dense inner wall (iw). Well preserved organelles are also clearly distinguishable including endoplasmic reticulum (er), Golgi stacks (g), mitochondria (m) and vacuoles (v). pm, plasma membrane. B-C, Immunogold labeling of (1→5)-α-L-arabinan epitopes with LM6. Gold particles (arrowheads) are mostly localized in the outer wall (ow) layer. In B, possible trans Golgi network (tgn) and secretory vesicles (sv) labeled with LM6. D, Immunogold labeling of fucosylated xyloglucan motif recognized by CCRC-M1 in the inner (iw) and outer wall (ow) layers. Note the presence of gold particles in vesicles in the vicinity of the plasma membrane (white arrow). E-F, Immunogold labeling of non fucosylated xyloglucan motif (XXXG) with LM15. Gold particles (arrowheads) are visible in the inner (iw) and mainly in the outer walls (ow). cw, cell wall; er, endoplasmic reticulum; g, Golgi stacks; iw, inner wall layer; m, mitochondria; ow, outer wall layer; pm, plasma membrane; sv, secretory vesicles; tgn, trans Golgi network; v, vacuoles. Scale bars = 1 µm (A), 0.5 µm (B, D, E) and 100 nm (C and F).

Figure 3 .

 3 Figure 3. Immuno-dot blot assay of pectin (oxalate extract) and hemicellulose (KOH extract) enriched fractions isolated from Arabidopsis pollen tube cell wall. Oxalate and KOH extracts (8, 4, 1 and 0.5 µg) were probed with JIM5 for partially methylesterified HG, LM5 for (1→4)-β-D-galactan, LM6 for (1→5)-α-L-arabinan and LM15 for non galactosylated XyG (XXXG). Controls (8 µg): a, arabinan from sugar beet; b, pectin with 8.6% methylesterification from citrus; c, XyG from tamarind seed; d, gum arabic from acacia.

Figure 4 .

 4 Figure 4. Analysis of Arabidopsis pollen tube XyG. A. MALDI-TOF mass spectrum of endo-glucanasegenerated XyG fragments from the cell wall of 16h-grown Arabidopsis pollen tubes. The structures of the XyG fragments are shown according to the nomenclature proposed by Fry et al. (1993). Underlined and bold structures represent O-acetylated side chains (+ 1 OAc). *, indicates the signal of XLFG fragment with K + adduct ion instead of Na + . B. Monosaccharide composition of 16h-grown Arabidopsis pollen tube cell wall before (untreated) and after (treated) endo-glucanase treatment. Only the monosaccharides composing XyG are shown: Fuc, fucose; Gal, galactose; Glc, glucose; Xyl, xylose. Others include arabinose, galacturonic acid, glucuronic acid, mannose and rhamnose.

Table I .

 I Summary of cytochemical staining and immunolocalization of cell wall polymers in in vitro grown Arabidopsis pollen tubes.

	Based on observations at fluorescent microscopy (FM) and electron microscopy (EM)
	levels.
	Target	Probe	Back from the tip	Tip
			FM	EM	FM
			level	level	level
	β-glucan	calcofluor white	+	na	+
	Callose	aniline blue	+	na	-
		anti-callose	nd	iw	nd
	Extensin	LM1	-	nd	-
	AGP	LM2	+/-	nd	+
		MAC207	+/-	nd	+
		JIM13	-	nd	-
	HG	JIM5	+	nd	+/-
		JIM7	+/-	nd	+
	Galactan	LM5	+/-	nd	+/-
	Arabinan	LM6	+	ow	+
		LM13	+	nd	+
	Xylogalactu-ronan	LM8	+	nd	+
	XyG	CCRC-M1	+	iw/ow	+
		LM15	+	iw/ow	+
	Controls	-	-	-	-
	AGP, arabinogalactan-protein; HG, homogalacturonan; XyG, xyloglucan. -, no labeling
	detected (at over 500 ms exposure time) to + labeling detected. na, not applicable; nd, not
	determined; iw, inner wall layer; ow, outer wall layer.

Table II .

 II Monosaccharide composition and linkage analysis of total pollen tube cell wall, pectin enriched (ammonium oxalate) and hemicellulose enriched (KOH) extracts.

		Pollen tube	Oxalate extract	KOH
		wall		extract
	NS (%)	-	78.5	97.6
	UA (%)	-	21.5	2.4
	Monosaccharide composition a
	Ara	43.6	24.7	23
		(± 4.5)	(± 1.9)	(± 7.4)
	Fuc	1.2	1.0	0.9
		(± 0.2)	(± 0.5)	(± 0.7)
	Gal	8.4	18.3	14
		(± 1.0)	(± 0.6)	(± 3.2)
	Glc	19.8	19.3	40.1
		(± 5.1)	(± 2.8)	(± 6.6)
	GalA	10.9	21.5	6.8
		(± 0.7)	(± 1.4)	(± 0.2)
	GlcA	2.2	3.4	1.5
		(± 3.0)	(± 0.8)	(± 0.4)
	Rha	5.3	4.2	2.4
		(± 0.8)	(± 0.2)	(± 0.7)
	Man	2.2	1.7	4.9
		(± 1.5)	(± 3.0)	(± 0.1)
	Xyl	6.2	5.8	6.5
		(± 1.2)	(± 0.6)	(± 0.4)
	Linkage analysis b
	t-Araf	-	27.4	8.5
	5-Araf	-	28.2	14.6
	2,5-Araf	-	3.7	2.2
	3,5-Araf	-	3.9	2.7
	t-Xyl	-	1.4	1.1
	2-Xyl	-	1	2.4
	t-Gal	-	1.7	6
	3-Gal	-	2	1.2
	4-Gal	-	1.5	nd
	6-Gal	-	4	1.8
	3,6-Gal	-	5.6	7.3
	4,6-Gal	-	1.2	2.2
	3-Glc	-	10	27.6
	4-Glc	-	8.4	15.4
	2,3-Glc	-	nd	7
	NS, neutral sugar; UA, uronic acid (determined by the phenol sulphuric and mHDP
	colorimetric assays). a Determined by GC and expressed as mol%. Ara, arabinose; Fuc, fucose;
	Gal, galactose; Glc, glucose; GalA, galacturonic acid; GlcA, glucuronic acid; Rha, rhamnose;
	Man, mannose; Xyl, xylose; nd, not detected. Values are the mean ± SD from two
	independent experiments. b Determined by GC-MS of partially methylated alditol acetates and
	expressed as % of total area of the identified peaks. t-Araf denotes 1,4-di-O-acetyl-1-deuterio-
	2,3,5-tri-O-methyl-D-arabinitol, etc…,.-not determined, nd. not detected.

Table III .

 III Relative quantification of XyG oligosaccharides obtained after endo-glucanase digestion of Arabidopsis pollen tube wall. Mass of the fragments [M+Na + ] + . b Hex, hexose; Pent, pentose; Dox, deoxyhexose; OAc, O-acetyl substituent. c Based on XyG oligosaccharide structures found in Pauly et al. (2001). d Values are means ± SD from MALDI spectra obtained after endo-glucanase digestion from three different Arabidopsis pollen tube cell wall extracts.

	Mass a	Composition b	Structure c	Relative abundance d (%)
	1085	Hex 4 Pent 3	XXXG	14.7 ± 3.3
	1247	Hex 5 Pent 3	XLXG/XXLG	5.7 ± 0.6
	1289	Hex 5 Pent 3 OAc 1	XLXG/XXLG + 1 OAc	27.6 ± 3.9
	1393	Hex 5 Pent 3 Dox 1	XXFG	8.4 ± 0.5
	1435	Hex 5 Pent 3 Dox 1 OAc 1	XXFG + 1 OAc	41.2 ± 6.6
	1555	Hex 6 Pent 3 Dox 1	XLFG	2.5 ± 0.1 e
	a

e Relative abundance of this oligosaccharide corresponds

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This work was supported by the University of Rouen (UR), le Grand Réseau de Recherche VATA de Haute Normandie and the CNRS. UR and VATA are also acknowledged for providing a doctoral fellowship (to FD).


Arnaud Lehner

Flavien Dardelle

Odile Soret-Morvan

Patrice Lerouge

Azeddine Driouich

Jean-Claude Mollet
email: jean-claude.mollet@univ-rouen.fr

Pectins in the cell wall of Arabidopsis thaliana pollen tube and pistil

Keywords: Arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tract AGP, arabinogalactan-protein, HG, Homogalacturonan, MAbs, monoclonal antibodies, PME, pectin methylesterase

Plant sexual reproduction involves the growth of tip-polarized pollen tubes through the female tissues in order to deliver the sperm nuclei to the egg cells. Despite the importance of this crucial step, little is known about the molecular mechanisms involved in this spatial and temporal control of the tube growth. In order to study this process and to characterize the structural composition of the extracellular matrix of the male gametophyte, immunocytochemical and biochemical analyses of Arabidopsis pollen tube wall have been carried out. Results showed a well defined localization of cell wall epitopes with highly esterified homogalacturonan and arabinogalactan-protein mainly in the tip region, weakly methylesterified homogalacturonan back from the tip and xyloglucan and (1→5)-α-L-arabinan all along the tube. Here, we present complementary data regarding 1) the ultrastructure of the pollen tube cell wall and 2) the immunolocalization of homogalacturonan and arabinan epitopes in 16h-old pollen tubes and in the stigma and the transmitting tract of the female organ. Discussion regarding the pattern of the distribution of the cell wall epitopes and the possible mechanisms of cell adhesion between the pollen tubes and the female tissues is provided.

Introduction

Fertilization of flowering plants requires the delivery of the two sperm cells, carried by the fast growing tip-polarized pollen tube, to the egg cell. At every stage of the pollen tube development within the stigma, style and ovary, pollen tubes are guided to the ovules via multiple signals that need to pass through the cell wall of the pollen tube to reach their targets. 1-6 The analysis of Arabidopsis pollen tube cell wall has recently been reported. [START_REF] Dardelle | Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall[END_REF] Results showed a well defined localization of cell wall epitopes with highly methylesterified homogalacturonan (HG)

and arabinogalactan-protein (AGP) mainly in the tip region, weakly methylesterified HG back from the tip and xyloglucan and arabinan all along the tube. In addition, according to the one letter nomenclature of xyloglucan 8

, the main motif of Arabidopsis pollen tube xyloglucan was XXFG harboring one O-acetyl group. In order to bring new information regarding the possible interaction between the pollen tubes and the female tissues, the ultrastructural organization of the pollen tube cell wall, the cytological staining and immunolocalization of the cell wall epitopes of the pistil and especially the transmitting tract (TT), a specialized tissue where pollen tubes grow, were carried out.

Scanning electron micrographs of pollen grain and pollen tube of Arabidopsis

Chemically fixed pollen tubes were observed with a SEM Zeiss EVO40 EP at 10 kV. The first two photographs show the change of shape of the pollen grain before and after imbibition in the liquid culture medium from an oblong to a spherical form (Fig. 1A,B). The pollen tube tip bulged from one of the three apertures (Fig. 1B) and a rapid polarized pollen tube cell elongation was observed (Fig. 1C).

One hour after the imbibition, pollen tube length reached about 30 µm (Fig. 1C). Pollen tube growth is accompanied by the periodic deposition of callose plugs which maintain the tube cell in the apical region of the tube (Fig. 1D). The surface of the pollen tube did not appear smooth but instead showed cell wall material apparently oriented slightly parallel to the direction of the tube elongation (Fig. 1E).

This contrasts with the view of the apical region of the pollen tube in which the cell wall showed a different organization (Fig. 1F).

Cell wall polysaccharides in pollen tube and pistil

Cytochemical staining and immunolocalization of cell wall epitopes in the pistil were carried out on 2 µm sections of flowers fixed with 4% formaldehyde and 1% glutaraldehyde, dehydrated in ethanol series and embedded in methacrylate resin. Samples were observed under Nomarski differential interference contrast optics or fluorescence illumination on a Leica DLMB microscope equipped with FITC (absorption, 485-520 nm; emission, 520-560 nm wavelength). Pictures were acquired with a Leica DFC300FX camera. Pollen tubes were grown in liquid medium for 16-h and labeled with monoclonal antibodies (MAbs) as previously described.

7

Weakly and highly methylesterified HGs were clearly detected along the whole tube with a weak (Fig. 2A) and a strong (Fig. 2B) labeling intensity at the tip, respectively. Cytochemical staining of carboxylated polysaccharides such as HG with toluidine blue (0.05% phosphate buffer pH 6.8) was performed on longitudinal sections of the pistil. Sections stained with toluidine blue showed a purple coloration of the tissues from the stigma, style, and ovary with a notably deeper staining within the TT and the ovules (Fig. 2C). Immunolocalization of weakly methylesterified HG with JIM5 exhibited a uniform labeling pattern over the different tissues (Fig. 2E). Immunolocalization of (1→5)-α-L-arabinan epitopes with LM6 showed a homogeneous fluorescence in all the tissues of the pistil except in the TT where the labeling appeared slightly denser (Fig. 2F) indicating the presence of branched rhamnogalacturonan-I (RG-I) and/or AGPs.

Discussion

Immuno-fluorescence of 16h-old pollen tubes with JIM5 and JIM7 showed similar labeling pattern seen with 6h-old pollen tubes [START_REF] Dardelle | Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall[END_REF] but with a tendency to fold during sample preparation because of the length of the pollen tubes. This labeling pattern is similar to the one observed with pollen tubes from other species such as potato, tobacco, petunia, jasmine and corn [START_REF] Fry | An unambiguous nomenclature for xyloglucan-derived oligosaccharides[END_REF][START_REF] Li | Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants[END_REF] with a dominant localization of the highly methylesterified HG at the tip and the weakly methylesterified HG epitopes behind the tip.

These results are consistent with the theoretical model of action of the Pectin Methyl Esterases (PMEs) during pollen tube growth. [START_REF] Bosch | Pectin methylesterase, a regulator of pollen tube growth[END_REF][START_REF] Geitmann | How to shape a cylinder: pollen tube as a model system for the generation of complex cellular geometry[END_REF] Indeed, variations in cell wall properties are likely to be related, at least in part, by the modulation of the level of methylesterification of HG. HGs are deposited in a highly methylesterified form in the tip region and demethylesterified by PMEs during the remodeling of the cell wall. The carboxyl groups of the HG, back from the tip, are able to complex calcium ions which could rigidify the cell wall. [START_REF] Geitmann | The architecture and properties of the pollen tube cell wall[END_REF] In addition, (1→5)-α-L-arabinans, side chains of RG-I, are also abundant in Arabidopsis pollen tube cell wall, both at the tip and behind the tip. [START_REF] Dardelle | Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall[END_REF] Arabinan side chains of RG-I are thought to prevent HG polymers from forming tight association [START_REF] Jones | Cell wall arabinan is essential for guard cell function[END_REF] and have been implicated in cell attachment. [START_REF] Iwai | Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells[END_REF][START_REF] Orfila | Altered middle lamella homogalacturonan and disrupted deposition of (1-5)-alpha-L-arabinan in the pericarp of Cnr, a ripening mutant of tomato[END_REF][START_REF] Peňa | Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell separation during prolonged storage of apple[END_REF] These polymers may have similar action during the pollen tube growth at the cell walls of both the pollen tube and the TT, also enriched in arabinans. Interestingly, arabinogalactan from the Trasmitting Tract Specific (TTS) AGP has been previously implicated in tobacco pollen tube growth and guidance. 17

Taken together, these results suggest a strong regulation of the degree of methylesterification of the HG in the pollen tube and the female tissue cell walls probably acting as a regulator of the rigidification of the cell wall which may regulate polarized cell growth. [START_REF] Bosch | Pectin methylesterase, a regulator of pollen tube growth[END_REF][START_REF] Geitmann | The architecture and properties of the pollen tube cell wall[END_REF] Similarly, changes in the degree of methylesterification of the HG in the TT cell wall may allow a local relaxation of the cell wall facilitating the intrusive growth of the pollen tubes in the extracellular matrix of this specialized tissue.

Cell adhesion domains can have mechanosensory properties in eukaryotic cells. [START_REF] Baluška | Cytoskeleton-plasma membranecell wall continuum in Plants. Emerging links revisited[END_REF] Adhesion between Arabidopsis pollen tubes and the TT cells has been observed and may be controlled by arabinans from AGPs [START_REF] Lennon | The structure of the transmitting tissue of Arabidopsis thaliana (L.) and the path of pollen tube growth[END_REF] and/or RG-I which might be an important cue for guiding the tube cells within the female tissue, as suggested with lily. [START_REF] Lord | Adhesion and guidance in compatible pollination[END_REF] Investigations on mutant pollen defective in arabinosyltransferases and PMEs or plants with abnormal TT should provide new insights into the function of pectic HG and arabinan side chains of RG-I in pollen tube growth and adhesion.

Figure 1 .

 1 Figure 1. Scanning electron micrographs of Arabidopsis pollen grains and pollen tubes during growth. A, dehydrated oblong pollen grains showing the pollen coat at the surface of the exine. B, pollen tube tip emerging from hydrated pollen grain. C, one hour-old pollen tube. D, view of a callose plug (arrowhead). E, surface of the pollen tube cell wall back from the apical zone. F, view of the pollen tube cell wall in the tip region. Scale bars = 10 µm (A, B, C and D) and 2 µm (E and F).

Figure 2 .

 2 Figure 2. Cytochemical staining and immunolocalization of cell wall epitopes in the pollen tube and pistil. (A-B), Immunolocalization of weakly and highly methylesterified HG with JIM5 and JIM7, respectively. (C) Immunolocalization of (1→5)-α-L-arabinan epitopes with LM6. (D), Cytochemical staining of acid pectins with toluidine blue on longitudinal sections of the pistil. (E, F), Immunofluorescence labeling of the cell wall polymer epitopes in longitudinal sections of the pistil with the MAbs JIM5 (E) and LM6 (F). Pollen tubes were grown for 16h in liquid medium. Pistils were dissected from closed flowers (i.e. before pollination). Scale bars = 20 µm (A, B), 10 µm (C) and 50 µm (D-F). o, ovule; ov, ovary; p, papillae; s, stigma; st, style; tt, transmitting tract.

Acknowledgements

The authors are thankful to R. Ngouala Finassi for his participation in pollen tube and pistil immunolocalization analyses. We are also grateful to P. Knox (University of Leeds, UK) for providing some of the monoclonal antibodies used in this study. This work was funded by the Grand Réseau de Recherche: Végétal, Agronomie et Transformation des Agroressources (VATA) de Haute Normandie, the University of Rouen (UR), and the CNRS.