Immunolocalization and possible roles of pectins during pollen growth and callose plug formation in angiosperms

To elucidate the possible roles of pectins during the growth of angiosperm pollen, we studied the distribution and changes in the properties of pectin in the pollen grains and tubes of Camellia japonica, Lilium longiflorum, and five other species at different growth stages by immunoelectron microscopy with monoclonal antibodies JIM5, against de-esterified pectin, and JIM7, against esterified pectin. We also studied the localization of arabinogalactan proteins, which are regarded as pectin-binding proteins, with monoclonal antibodies JIM13 and LM2, against arabinogalactan proteins. Similar results were obtained for all species: JIM5 labeled the intine and part of the callose layer in germinated pollen grains, and labeled the outer layer of the tube wall, the Golgi vesicles, and the callose plug in the pollen germinated in vitro, but did not label any part of immature pollen grains. In contrast, JIM7 labeled the intine of both immature and mature pollen grains, labeled the Golgi vesicles around the Golgi bodies, and strongly labeled the outer layer of the cell wall and the Golgi vesicles in the tube tip region. On the other hand, the distribution of arabinogalactan proteins detected with JIM13 was different for each species, and does not suggest a close relationship between pectin and arabinogalactan proteins. LM2 scarcely reacted with the specimens. We discuss the contribution of pectins to tube wall formation and fertilization and deduce a mechanism of callose plug formation.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

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

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

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

Exploding pollen in Montrichardia arborescens (Araceae)

Pollen grains of Montrichardia are inaperturate with psilate ornamentation. The pollen wall is formed by a thin ectexine and an extraordinarily thick intine. In living as well as in dead pollen grains contact with water leads to a rapid swelling of the intine followed by an explosive opening of the exine. Within a few seconds a thick tube is formed, which is not the pollen tube. The pollen protoplast is situated at the tip of the tube. These intine tubes are interpreted as pollen connecting tools to keep pollen grains together and adhere them to the cuticle of the hairless pollinators.     

Lead Stress Disrupts the Cytoskeleton Organization and Cell Wall Construction During <Emphasis Type="Italic">Picea wilsonii</Emphasis> Pollen Germination and Tube Growth

Lead is a widespread pollutant and has been reported to inhibit pollen tube development, but the mechanism of toxicity involved remains unclear. Here, we report that lead stress significantly prevented Picea wilsonii pollen germination and tube growth and also dramatically altered the tube morphology in a concentration-dependent manner. Fluorescence labeling with JIM 5 (anti-acidic pectin antibody) and Calcofluor white revealed the lead-induced decline of acidic pectin and cellulose, especially in the subapical region. Decolorized aniline blue staining showed the marked accumulation of callose in the apical and subapical regions of lead-treated tubes. Fluorescence labeling with Alexa Fluor 568 phalloidin and anti-tubulin antibody revealed that the distribution of the cytoskeleton in P. wilsonii pollen grains and tubes were developmentally regulated and that lead disturbed the cytoskeleton organization, especially in the shank of the pollen tubes. Taken together, our experiments revealed a link between the dynamics of cytoskeleton organization and the process of P. wilsonii pollen tube development and also indicated that lead disturbed the cytoskeleton assembly and, consequently, cell wall construction. These findings provide new insights into the mechanism of lead toxicity in the tip growth of pollen tubes.     

内钙拮抗剂TMB-8对白皮松花粉管细胞壁构建的影响

应用荧光显微技术、激光共聚焦扫描显微技术、单克隆抗体免疫荧光标记技术以及傅里叶变换显微红外光谱分析(FTIR)等手段,研究了内钙拮抗剂TMB-8对白皮松花粉管胞内Ca2+分布、花粉管生长以及细胞肇构建等的影响.结果表明,白皮松花粉管经TMB-8处理后,胞内的Ca2+浓度下降,花粉管内典型的Ca2+浓度梯度消失,花粉萌发...     

Immunocytochemical localization of pectins in the maturing anther of Allium cepa L.

Distribution of pectins in cell walls of maturing anther of Allium cepa L. was investigated. The monoclonal antibodies against defined epitopes of pectin were used: JIM5 recognizing unesterified pectin and JIM7 recognizing esterified pectin. It has been found that the cell walls of all anther tissues mainly contain esterified pectins. In the somatic tissues only small amounts of unesterified pectins are present in the cell wall junctions and adjacent middle lamellae and in the cell walls of the connective tissue. Thickening of the epiderm cell walls and growth of trabeculae in endothecium are completed through deposition of esterified pectins. In the cell walls of the middle layer and tapetum, unesterified pectins have been found only prior to their disintegration. The primary wall of microsporocytes is made up mainly of esterified pectins. Unesterified pectins occur outside microsporocytes only prior to the callose isolation stage. The presence of esterified pectins has also been detected on the surface of the callose wall surrounding dividing microsporocytes. Lysis of those pectins takes place after microsporogenesis, simultaneously with the lysis of the callosic walls. Before these processes pectins are unesterified. In the sporoderm of pollen grains mainly esterified pectins occur. They have been localized in the intine and aperture. The level of unesterified pectins in the intine is markedly lower.     

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

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

Isolation of de-exined pollen and cytological studies of the pollen intines of Pinus bungeana Zucc. Ex Endl. and Picea wilsonii Mast

To study the cytological and biochemical characteristics of intine, pollen deprived of exine, or de-exined pollen, was isolated from the gymnosperms Pinus bungeana and Picea wilsonii. The factors influencing the isolation rate were examined. Cellulose, callose, pectin, and arabinogalactan proteins (AGPs) were localized in this material using fluorescent probes, and components of the isolated intine were further analyzed by Fourier transform infrared (FTIR) microspectroscopy. The isolation protocol was repeatable and reliable. Cellulose was found to be evenly distributed on the surface of the intine, as indicated by strong calcofluor White ST (CW) fluorescence, and aniline blue staining revealed that callose was present on the intine of P. bungeana but not on that of P. wilsonii. Immunolabeling revealed that acidic pectin epitopes recognized by the monoclonal antibody JIM5 were present on the pollen intine, as well as esterified pectin recognized by the monoclonal antibody JIM7, and AGPs recognized by the LM2 antibody. Two lectin binding sites, the concanavalin agglutinin (Con A) and soybean agglutinin (SBA) binding sites, were present on the intine surface, but no wheat germ agglutinin (WGA) binding sites were detectable. These results were confirmed by FTIR analysis.     

Ultrastructural immunolocalization of periodic pectin depositions in the cell wall ofNicotiana tabacum pollen tubes

Summary The monoclonal antibody (MAb) JIM5, marking acidic pectins, was used to localize ultrastructurally pectin molecules in the pollen tube wall ofNicotiana tabacum. Longitudinal sections of LR-White embedded pollen tubes were exposed to antibody treatment; accumulations of pectins were identified by counting the density of the gold particles representing the pectin epitopes along the pollen tube wall. Significant accumulations of gold grains were marked and the distances between them were measured. In many pollen tubes a more or less regular distribution of the accumulations was observed along the tube indicating a periodical deposition of pectin. The distances between the accumulations were 4–6 m. Most of the label was found in the inner part of the outer layer of the bilayered cell wall. These findings correspond to and confirm the earlier observation by our group reporting ring-shaped periodical deposits in pollen tubes after immunofluorescence labelling with the MAb JIM5 under the confocal laser scanning microscope.Abbreviations Ab antibody - MAb monoclonal antibody     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

Pectin secretion and distribution in the anther during pollen development in Lilium

Using the monoclonal antibodies JIM 5 and 7, pectin was immunolocalized and quantitatively assayed in three anther compartments of Lilium hybrida during pollen development. Pectin levels in both the anther wall and the loculus increased following meiosis, were maximal during the early microspore stages and declined during the remainder of pollen ontogenesis. In the microspores/pollen grains, pectin was detectable at low levels during the microspore stages but accumulated significantly during pollen maturation. During early microspore vacuolation, esterified pectin epitopes were detected both in the tapetum cytoplasm and vacuoles. In the anther loculus, the same epitopes were located simultaneously in undulations of the plasma membrane and in the locular fluid. At the end of microspore vacuolation, esterified pectin epitopes were present within the lipids of the pollenkitt, and released in the loculus at pollen mitosis. Unesterified pectin epitopes were hardly detectable in the cytoplasm of the young microspore but were as abundant in the primexine matrix as in the loculus. During pollen maturation, both unesterified and esterified pectin labelling accumulated in the cytoplasm of the vegetative cell, concurrently with starch degradation. In the mature pollen grain, unesterified pectin epitopes were located in the proximal intine whereas esterified pectin epitopes were deposited in the distal intine. These data suggest that during early microspore development, the tapetum secretes pectin, which is transferred to the primexine matrix via the locular fluid. Further, pectin is demonstrated to constitute a significant component of the pollen carbohydrate reserves in the mature grain of Lilium. Received: 3 July 2000 / Accepted: 19 October 2000     

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

Background and Aims

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

Methods

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

Key Results

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

Conclusions

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

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

鹅掌楸属植物花粉萌发前后壁的超微结构

观察描述了在电镜下中国鹅掌楸（Ｌｉｒｉｏｄｅｎｄｒｏｎｃｈｉｎｅｎｓｅ）和北美鹅掌楸（Ｌ．ｔｕｌｉｐｉｆｅｒａ）２种植物花粉壁的超微结构及其水合后的变化。（１）成熟花粉壁由６层组成,即外壁３层──外层,中层１和中层２,内壁３层──内壁１,内壁２和内壁３。（２）花粉水合时,在内壁３与质膜之间由Ｐ一粒子（多糖－粒子）和被膜小泡参与形成新层。（３）花粉萌发时,由内壁３的一部分和新层突出萌发孔共同形成花粉管壁。（４）新层于花粉管形成早期分成２层──外染色深的果胶层和内电子透明的胼胝质层。     

Immunocytochemical localization of callose in the germinated pollen ofCamellia japonica

Summary A polyclonal antibody against -1,3-glucan, callose, extracted from the pollen tube wall ofCamellia japonica was raised in mice and, using it as a probe, the localization of callose in the germinated pollen was studied. By confocal laser scanning microscopy, callose was found in the tip region of the pollen tube and the tube wall; the immuno-fluorescence in the tube wall was less toward the base of the tube. In contrast, the tip region did not fluoresce although the whole of the tube wall did strongly with aniline blue, the specific dye for callose. Immuno-electron microscopy showed that callose was also found in Golgi vesicles which concentrated in the tip region of the pollen tube, the inner layer of the tube wall, callose plugs, and Golgi vesicles in the pollen grain. Immuno-gold labeling was often detected on the fibrous structures in Golgi vesicles and callose plugs. Based on these results, the participation of Golgi vesicles in the formation of the tube wall and callose plugs was discussed.Abbreviation TBS Tris-buffered saline - Tris Tris(hydroxy-methyl)-aminomethane - PBS phosphate-buffered saline - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - CLSM confocal laser scanning microscopy - DP degree of polymerization     

Lily (<Emphasis Type="Italic">Lilium longiflorum</Emphasis> L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.     

2, 6-dichlorobenzonitrile Causes Multiple Effects on Pollen Tube Growth beyond Altering Cellulose Synthesis in Pinus bungeana Zucc

Cellulose is an important component of cell wall, yet its location and function in pollen tubes remain speculative. In this paper, we studied the role of cellulose synthesis in pollen tube elongation in Pinus bungeana Zucc. by using the specific inhibitor, 2, 6-dichlorobenzonitrile (DCB). In the presence of DCB, the growth rate and morphology of pollen tubes were distinctly changed. The organization of cytoskeleton and vesicle trafficking were also disturbed. Ultrastructure of pollen tubes treated with DCB was characterized by the loose tube wall and damaged organelles. DCB treatment induced distinct changes in tube wall components. Fluorescence labeling results showed that callose, and acidic pectin accumulated in the tip regions, whereas there was less cellulose when treated with DCB. These results were confirmed by FTIR microspectroscopic analysis. In summary, our findings showed that inhibition of cellulose synthesis by DCB affected the organization of cytoskeleton and vesicle trafficking in pollen tubes, and induced changes in the tube wall chemical composition in a dose-dependent manner. These results confirm that cellulose is involved in the establishment of growth direction of pollen tubes, and plays important role in the cell wall construction during pollen tube development despite its lower quantity.