金鱼草花粉管亚原生质体的分离及在培养中的行为

应用酶法从金鱼草花粉管中分离出大量的亚原生质体。这种亚原生质体培养在 D_2液体培养基中,不论是有核的或是无核的都能再生厚的细胞壁和生长出花粉管状的管状结构。这些管状结构除了它们的顶端区外也沉积厚的细胞壁。随着管状结构的生长,内含物逐渐移向管状结构的顶端。当生长停止后,内含物可能完全被耗尽;有时管状结构的顶端破裂,内含物释放至培养液中。无核和有核亚原生质体同样显示有正常花粉管的基因表达的特性,即在培养中有类似花粉管生长的行为。这一事实表明在萌发的花粉管中有预先合成的 mRNA 的存在。     

Development of subprotoplasts from in vitro-grown tobacco pollen tubes

Summary Plasmolyzed pollen tubes of Nicotiana tabacum each released one to three subprotoplasts from their tips when treated with wall-degrading enzymes. Wall regeneration and the further development of the subprotoplasts were studied by both light and electron microscopy. Karyoplasts and cytoplasts incubated in a poor culture medium regenerated a cell wall within 60 min; cellulose microfibrils and callose were shown to be present. In 30%–40% of the cells, one-third of which were nucleate, cell chains and tubes developed by polar growth in a ratio of about 11. They sometimes reached a length 7–9 times the diameter of the former subprotoplast within 5 h of incubation. With longer incubation periods the cell wall became two-layered, its ultrastructure resembling that of the pollen tubes. The capacity of cytoplasts to regenerate a wall and develop cell chains and tubes can be explained by the properties ascribed to the cytoplasm of pollen tubes.Extended version of part of a contribution (poster) presented at the 14th International Botanical Congress, Berlin (West), July 1987.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

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     

Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

Cytochemical localization of some hydrolases in the pollen and pollen tubes of Amaryllis vittata Ait

Some hydrolases are localized cytochemically in the pollen and pollen tubes of Amaryllis vittata Ait. The function of different enzymes is discussed in relation to pollen tubes morphogenesis. Activity of most of the enzymes was confined to colpus region, pollen wall and general cytoplasm of pollen and pollen tube. The activity of hydrolytic enzymes like acid monophosphoesterase and lipase and was nil in the exine of both germinated and ungerminated pollen, whereas intense reaction for esterase was observed in exine. Enzyme activity increased after germination which suggest the hydrolysis of stored metabolites and synthesis of proteins and other metabolites for the active growth of pollen tube. Intense reaction for enzymes like alkaline phosphomonoesterase, ATP-ase, 5-nucleotidase etc. at the tip region of pollen tube suggest their role in physiological processes associated with exchange of materials through intercellular transport during tube wall polysaccharide biogenesis.     

Division in Isolated Generative Cells of Allemanda neriilolia

An osmotic shock method of isolating generative cells from Allemanda neriifolia was described. Fresh pollen grains were first placed ill a Brewbaker and Kwack's medium (BK medium) containing 50% sucrose, incubated at 28℃ for 2 hours. During this incubation period pollen grains germinated and produced pollen tubes measuring about 200 μm long. After this initial incubation period, a fixed amount of BK medium without sucrose was added thus diluting the original medium to a sucrose concentration of 30% – an optimum concentration for generative cell growth. The addition, of the BK medium without sucrose brought about an osmotic shock effect on the pollen tubes and caused most of the tubes to burst at the tip region thus releasing the contents together with the generative cell from the tube into the 30% sucrose + BK medium. After isolation and filtering into a fresh lot of 30% sucrose + BK medium, generative cells changed from spindle into spherical-shaped cells. In the 30% sucrose + BK medium, the generative cells divided and within a short period of 3 to 5 hours a laege number of cells at various stages of mitosis was obtained.     

Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes

The behavior and role of the microtubule (MT) and actin-myosin components of the cytoskeleton during pollen tube growth in two species of Pinus were studied using anti--tubulin, rhodamine-phalloidin, anti-myosin, and the appropriate inhibitors. Within germinated pollen tubes MTs were arranged obliquely or transversely, but in elongated tubes they were arranged along the tube's long axis. MTs were localized in the tube tip region, excluding the basal part. Altered growth was found in pollen tubes treated with colchicine; the tips of many pollen tubes incubated in the liquid medium were branched and/or rounded, and those in the agar medium were divided into many branches. Both the branching and the rounding were considered to be caused by the disturbance of polarizing growth of the tube due to MT disorganization with colchicine treatment. Actin filaments (F-actin) were found in the major parts of many pollen tubes along their long axis, excluding the tip region. In a few tubes, however, F-actin was distributed throughout the tube. The areas in the pollen tube containing F-actin were filled with abundant cytoplasmic granules, but the areas without F-actin had very few granules. The tube nucleus, which migrated from the grain area into the tube, was closely associated with F-actin. Germination of pollen grains treated with cytochalasin B was little affected, but further tube elongation was inhibited. Myosin was identified on cytoplasmic granules and to a lesser extent on the tube nucleus in the pollen tubes. Several granules were attached to the nuclear envelope. Tube growth was completely inhibited by N-ethylmaleimide treatment. In generative cells that were retained in the pollen grain, both MT and F-actin networks were observed. Myosin was localized on the cytoplasmic granules but not on the cell surface. In conclusion, it was shown that actin-myosin and MTs were present in gymnospermous Pinus pollen tubes and it is suggested that the former contributed to outgrowth of the tubes and the latter contributed to polarized growth. Several differences in the behavior of cytoskeletal elements in generative cells compared to angiosperms were revealed and are discussed.     

Apical F‐actin‐regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall

In tip‐confined growing pollen tubes, delivery of newly synthesized cell wall materials to the rapidly expanding apical surface requires spatial organization and temporal regulation of the apical F‐actin filament and exocytosis. In this study, we demonstrate that apical F‐actin is essential for the rigidity and construction of the pollen tube cell wall by regulating exocytosis of Nicotiana tabacum pectin methylesterase (NtPPME1). Wortmannin disrupts the spatial organization of apical F‐actin in the pollen tube tip and inhibits polar targeting of NtPPME1, which subsequently alters the rigidity and pectic composition of the pollen tube cell wall, finally causing growth arrest of the pollen tube. In addition to mechanistically linking cell wall construction and apical F‐actin, wortmannin can be used as a useful tool for studying endomembrane trafficking and cytoskeletal organization in pollen tubes.     

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract

In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.     

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     

The wall ofPinus sylvestris L. pollen tubes

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

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.     

Isolation and Early in Vitro Development of Young Pollen Protoplasts in Nicotiana tabacum

For isolating young pollen protoplasts in Nicotiana tabacum. The authors had established two efficient enzymatic methods via anther preculture or pollen starvation pretreatment. Procedure of the first method included the following steps: 1. Cold pretreatment of flower buds with pollen at late unicellular to early bicellular stage; 2. Anther floating culture for pollen shedding into the culture medium followed by dehiscence of exine; 3. Enzymatic maceration of exine-dehisced pollen resulting in degradation of intine and release of pollen protoplasts in large quantity. Procedure of the second method involved the following steps: 1. Culture of pollen at middle bicellular in Kyo and Harada' B medium for starvation: 2. Enzymatic maceration of starvated pollen resulting in release of pollen protoplasts and subprotoplasts. Factors affecting the results of both methods as well as early in vitro developmental events of young pollen protoplasts were studied. The protoplasts could be induced either to trigger the first sporophytic division or to continue the gametophytic pathway leading germinatation of pollen tubes !ndicating their potentiality of inducing both sporophytic and gametophytic development of pathway. In rare instance a quite interesting phenomenon was observed that a pollen protoplast first divided into two daughter cells and one of which then germinated a pollen tube. It may insinuate that such pollen protoplasts initially induced a sporophytic pathway could reverse induce a gametophytic pathway.     

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.     

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.     

Calcium ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum

The effects of the calcium inonophore A 23187 on growing pollen tubes of Lilium longiflorum Thunb. cv. Ace were investigated with the light and electron microscope. Tip growth is slowed down and stopped within 20 min after application of 5x10^-5 M ionophore A 23187. The main effects are the disappearance of the clear zone at the pollen tube tip and a thickening of the cell wall at the tip and at the pollen tube flanks. This effect on cell wall formation is confirmed under the electron microscope: The vesicular zone in treated pollen tubes is reduced, numerous vesicular contents are irregularly integrated in the pollen tube wall not only in the tip, but over a long distance of the pollen tube wall. In addition, effects on mitochondria and dictyosomes are observed. These results are interpreted as a disorientation of the Ca²⁺-based orientation mechanism of exocytosis after equilibration of the Ca²⁺-gradient     

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

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 16-h-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.Key words: arabinan, cell adhesion, cell wall, homogalacturonan, pistil, pollen tube growth, transmitting tractFertilization 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.^1–6The analysis of Arabidopsis pollen tube cell wall has recently been reported.⁷ 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.