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     

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.     

Immuno-gold localization of α-L-arabinofuranosyl residues in pollen tubes of Nicotiana alata Link et otto

Immuno-gold labelling using a monoclonal antibody (PCBC3) with a primary specificity for -L-arabinofuranosyl residues was used to locate these residues in pollen tubes of Nicotiana alata grown in vivo. The antibody bound to the outer fibrillar layer of the pollen-tube wall: the inner, non-fibrillar wall layer was not labelled. Cytoplasmic vesicles (0.2 m diameter) were also labelled. The antibody may bind to an arabinan in the pollen-tube wall.     

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     

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     

Arabinogalactan proteins at the cell surface ofBrassica sperm andLilium sperm and generative cells

Antibodies to arabinogalactan proteins were tested for binding to sperm cells ofBrassica campestris and to generative cells and sperm ofLilium longiflorum. Two monoclonal antibodies, JIM8 and JIM13, bound toBrassica sperm in pollen grains and pollen tubes and to isolated sperm. Sperm pairs retained within the vegetative cell inner plasma membrane fluoresced more brightly than single sperm, indicating that the vegetative cell inner plasma membrane that surrounds sperm pairs also contains arabinogalactan proteins. Isolated sperm pairs exhibited a uniform fluorescence while single sperm had patches of fluorescence. InLilium, isolated generative cells and single sperm cells bound antibodies in a patchy pattern. Antibodies to arabinogalactan proteins may be useful in describing the overall shape of sperm cells and for identifying sperm among other cell types.     

Localization of arabinogalactan proteins in anther,pollen, and pollen tube of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L.

Summary. Western blot analysis indicated the presence of two epitopes recognized by the anti-arabinogalactan protein antibodies JIM13 and LM2 and the absence of the JIM4 epitope in mature tobacco anthers. Immunoenzyme localization of arabinogalactan proteins (AGPs) with JIM13 showed that AGPs accumulate mainly at the early stages of anther development. AGP content and distribution were also investigated at the ultrastructural level in pollen tubes grown in vivo and in vitro. Abundant AGPs were present in the transmitting tissue of styles, and the AGP content of the extracellular matrix changed during pollen tube growth. In pollen tubes, immunogold particles were mainly distributed in the cell wall and cytoplasm, especially around the peripheral region of the generative-cell wall. β-D-Glucosyl Yariv reagent, which specifically binds to AGPs, caused slow growth of pollen tubes and reduced immunogold labeling of AGPs with JIM13 in vitro. These data suggest that AGPs participate in male gametogenesis and pollen tube growth and may be important surface molecules in generative and sperm cells. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Morphological and histochemical differentiation of the pollen wall ofBetula pendula Roth,during dormancy up to anthesis

Summary Anthers ofBetula pendula were collected at regular intervals during the dormancy period until anthesis. Ultrathin sections of maturing pollen grains were especially stained for polysaccharides and proteins and examined with TEM to determine whether structural or/and chemical changes in the pollen wall occur during the dormancy period of the plant life cycle. At the beginning of the dormancy period, the microspore wall consists of a well developed tectum, columellae and a foot layer. Spinules and supratectal elements are prominent. Microchannels are present in the tectum but not obvious in the foot layer. Some of the columellae are not clearly connected with the foot layer, but some connections are evident. Pores are filled with a thick fibrillar network flocculent material. The cytoplasm is packed with starch grains and lipid globules. The stainability for acidic and neutral polysaccharides and protein was tested, and variations in the pollen wall are illustrated. As temperature increased towards the end of dormancy and before anthesis there is obvious differentiation in the morphology of the pollen wall. The granular fibrillar layer beneath the pore and the Zwischenkörper are the most variable part of the wall. Different histochemical reactions observed in different layers at the aperture sites indicate different functions of these layers.     

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     

Ultrastructure of the sperms ofPlumbago zeylanica

Summary Male gametes ofPlumbago zeylanica were examined in pollen grains and tubes using light and electron microscopy of chemically and physically fixed tissues, and Nomarski interference microscopy of isolated, living sperm cells. Male gametes are elongate, spindleshaped cells containing a nucleus, mitochondria, ER, ribosomes, vesicles, dictyosomes, probable microfilaments, and a variable number of plastids. In mature pollen grains ofP. zeylanica, the two sperm cells are directly linked; they share a transverse cell wall with plasmodesmata and are enclosed together by the inner vegetative cell plasma membrane. One of these two sperms is also associated with the vegetative nucleus as a consistent feature of pollen grain organization. The basis of this association appears to be a long, narrow projection of the sperm cell (averaging < 1 m wide and about 30 m long) which wraps around the periphery of the vegetative nucleus and occupies embayments of that nucleus. This association is maintained throughout pollen tube growth but becomes less extensive near the completion of tube growth and is severed following tube discharge. The consistent occurrence of the sperm-vegetative nucleus association in pollen grains, tubes and isolated pollen cytoplasm suggests that the two structures may be directly connected, but attempts to visualize this type of connection were unsuccessful. Possibly, the entwining nature and extent of complementary interfaces between vegetative nucleus and sperm may have a role in stabilizing their association. Functionally, the two sperms and vegetative nucleus appear to travel as a linked unit within the pollen tube, possibly increasing the effectiveness of gamete delivery and helping to ensure nearly simultaneous transmission of sperms into the receptive megagametophyte.     

Immunocytochemical and chemical analyses of Golgi vesicles isolated from the germinated pollen ofCamellia japonica

As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically. The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose, arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall.     

Requirements for division of the generative nucleus in cultured pollen tubes ofNicotiana

Summary Production of sperm cells by division of the generative cell occurs during growth ofNicotiana (tobacco) pollen tubes through the sporophytic tissue of the style, and is associated with transition to the second phase of pollen-tube growth. WhenNicotiana pollen tubes are grown in liquid culture, the extent of generative-nucleus division and the timing of this division depend on the chemical composition of the medium. Addition of reduced forms of nitrogen, either as mixed amino-acids (0.03% w/v of an acid hydrolysate of casein) or as 1 mM ammonium chloride, induces division of the generative nucleus in over 90% of the tubes; 3 mM calcium nitrate does not stimulate division. Individual amino-acids differ in their ability to induce this division. Contaminants in some batches of poly(ethylene glycol), which is a major component of pollen-tube growth media, inhibit generative-nucleus division; this inhibition is greater in the absence of nitrogen, which increases the observed nitrogen-dependence of division. Reduced forms of nitrogen are also required for growth of pollen tubes after division, when callose plugs are deposited. In the absence of nitrogen, growth continues until the point where sperm cell production would normally occur, then ceases. Addition of amino-acids or ammonium chloride thus allows cultured pollen tubes ofNicotiana to progress to their second phase of growth. WhenNicotiana pollen is germinated in a complete culture medium at 25–26°C, sperm nuclei are first observed in the growing tubes after about 10 h, and by about 16 h most of the tubes have undergone division; at lower temperatures, division is delayed. The timing of division also varies between species ofNicotiana, but division occurs similarly in self-compatible and self-incompatible species. Anaphase in an individual pollen tube is calculated to take less than 4 min. The resultant sperm nuclei usually trail behind the vegetative nucleus, but a variety of arrangements of the three nuclei are observed.Abbreviations DAPI 4,6-diamidino-2-phenylindole - PEG poly(ethylene glycol) - OG ordinary grade of PEG - SP Specially Purified for Biochemistry grade of PEG     

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.     

Cell surface arabinogalactan-proteins and their relation to cell proliferation and viability

Summary We have used high-pressure freezing followed by freeze substitution (HPF/FS) to preserve in vivo grown lily pollen tubes isolated from the style. The results indicated that HPF/FS (i) allows excellent preservation of the pollen tubes, (ii) maintains in situ the stylar matrix secreted by the transmitting tract cells, and (iii) preserves the interactions that exist between pollen tubes. Particular attention has been given to the structure of the pollen tube cell wall and the zone of adhesion. The cell wall is composed of an outer fibrillar layer and an inner layer of material similar in texture and nature to the stylar matrix and that is not callose. The stylar matrix labels strongly for arabinogalactan proteins (AGPs) recognized by monoclonal antibody JIM13. The zone of adhesion between pollen tubes contains distinct matrix components that are not recognized by JIM13, and apparent cross-links between the two cell walls. This study indicates that HPF/FS can be used successfully to preserve in vivo grown pollen tubes for ultrastructural investigations as well as characterization of the interactions between pollen tubes and the stylar matrix.Abbreviations AGPs arabinogalactan proteins - FS freeze substitution - HPF high-pressure freezing     

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     

Effects of pollen selection on progeny vigor in a Cucurbita pepo x C. texana hybrid

We examined the effects of pollen selection for rapid pollen-tube growth on progeny vigor. First, we crossed a wild gourd (Cucurbita texana) to a cultivated zucchini (Cucurbita pepo cv Black Beauty) to produce an F₁ and then an F₂ generation. Half of the F₁ seeds were produced by depositing small loads of C. texana pollen onto the stigmas of C. pepo. These small pollen loads were insufficient to produce a full complement of seeds and, consequently, both the fast- and the slow-growing pollen tubes were permitted to achieve fertilization. An F₂ generation was then produced by depositing small loads of F₁ pollen onto stigmas of F₁ plants. The F₂ seeds resulting from two generations of small pollen loads are termed the non-selected line because there was little or no selection for pollen-tube growth rate on these plants. The other half of the F₁ and F₂ seeds were produced by depositing large pollen loads (>10 000 pollen grains) onto stigmas and then allowing only the first 1% or so of the pollen tubes that entered the ovary to fertilize the ovules. We did this by excising the styles at the ovary at 12–15 h after pollination. The resulting F₂ seeds are termed the selected line because they were produced by two generations of selection for only the fastest growing pollen tubes. Small pollen loads from the F₂plants, both the selected and the non-selected lines, were then deposited onto stigmas of different C. pepo flowers, and the vigor of the resulting seeds was compared under greenhouse and field conditions. The results showed that the seeds fertilized by pollen from the selected line had greater vegetative vigor as seedlings and greater flower and fruit production as mature plants than the seeds fertilized by pollen from the non-selected line. This study demonstrates that selection for fast pollen-tube growth (selection on the microgametophyte) leads to a correlated increase in sporophyte (progeny) vigor.     

Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts

Summary Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu_(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 M CuSO₄ did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu²⁺ concentration under these conditions was about 1.0 M due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 M added CuSO₄ when NH₄Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 M CuSO₄, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 m/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO₄ from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO₄ (300 M) had a similar morphogenetic effect to CuSO₄, but addition of 300 M L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe_(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO₄ and FeSO₄. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO₄ or FeSO₄, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO₄ suggests that Cu_(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.Abbreviations A₄₇₅ absorbance at 475 nm - DAPI 4,6-diamidino-2-phenylindole - EDTA ethylene-diamine N,N,N,N-tetraacetic acid - MES 2-(N-morpholino)-ethane sulphonic acid - OG ordinary grade of poly(ethylene glycol) - PEG poly(ethylene glycol) - SP Specially Purified for Biochemistry grade of poly(ethylene glycol)     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B