Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

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     

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms, pollen tube growth is critical for double fertilization and seed formation. Many of the factors involved in pollen tube tip growth are unknown. Here, we report the roles of pollen-specific GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE-LIKE (GDPD-LIKE) genes in pollen tube tip growth. Arabidopsis thaliana GDPD-LIKE6 (AtGDPDL6) and AtGDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein (GFP)-AtGDPDL6 and GFP-AtGDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes. Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with AtGDPDL6 or AtGDPDL7. This sterility was associated with defective male gametophytic transmission. Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo, consistent with the thin and fragile walls in their tips. Cellulose deposition was greatly reduced along the mutant pollen tube tip walls, and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1 (CSLD1) and CSLD4 was impaired to the apex of mutant pollen tubes. A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth, suggesting that members of this family have conserved functions in angiosperms. Thus, pollen-specific GDPD-LIKEs mediate pollen tube tip growth, possibly by modulating cellulose deposition in pollen tube walls.     

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.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.     

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     

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

Arabidopsis VPS18 plays an important role in regulating pollen tube growth through mediating the late endocytic trafficking and secretion of pectin and associated enzymes to the cell wall.     

Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins

Summary Monoclonal antibody PCBC3, raised against stylar extracts fromNicotians, alata flowers, was deduced from enzyme-linked immunosorbent assays and inhibition of immuno-gold labelling on tissue sections to bind specifically to carbohydrate epitopes on arabinogalactan proteins (AGPs) but not to other arabinose-containing cell wall polysaccharides. When pollen grains ofN. tabacum were hydrated in fixative, PCBC3 bound to vesicles in the vicinity of the endoplasmic reticulum but, when grains were hydrated for 20 min in culture medium before fixation, binding was restricted to the plasma membrane. The generative-cell plasma membrane was also labelled in grains ofLycopersicon peruvianum. In pollen tubes ofN. tabacum grown in liquid culture, the AGPs detected by PCBC3 were located in several regions, including the plasma membrane, tubular-vesicular structures (plasmalemmasomes) at and under the plasma membrane, and multilamellar bodies within vacuoles, features generally associated with endocytosis. Labelling was not evident in secretory vesicles or the plasma membrane at the pollen-tube tip. The AGPs detected with PCBC3 were also present in pollen-tube walls, near the interface between the inner, callosic layer and the outer, fibrillar, pectic layer. Pollen tubes ofN. tabacum grown in medium lacking added CuSO₄ produce a wall with an abnormally thickened fibrillar layer, and this layer was uniformly labelled with PCBC3. The disposition of wall AGPs thus changes in pollen tubes of different morphologies.Abbreviations AGP arabinogalactan protein - -L-Araf -L-arabinofuranose - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - PBS phosphate-buffered saline     

Localization of pectins and arabinogalactan-proteins in lily (Lilium longiflorum L.) pollen tube and style, and their possible roles in pollination

In lily, adhesion of the pollen tube to the transmitting-tract epidermal cells (TTEs) is purported to facilitate the effective movement of the tube cell to the ovary. In this study, we examine the components of the extracellular matrices (ECMs) of the lily pollen tubes and TTEs that may be involved in this adhesion event. Several monoclonal antibodies to plant cell wall components such as esterified pectins, unesterified pectins, and arabinogalactan-proteins (AGPs) were used to localize these molecules in the lily pollen tube and style at both light microscope (LM) and transmission electron microscope (TEM) levels. In addition, (-d-Glc)₃ Yariv reagent which binds to AGPs was used to detect AGPs in the pollen tube and style. At the LM level, unesterified pectins were localized to the entire wall in in-vivo- and in-vitro-grown pollen tubes as well as to the surface of the stylar TTEs. Esterified pectins occurred at the tube tip region (with some differences in extent in in-vivo versus in-vitro tubes) and were evenly distributed in the entire style. At the TEM level, esterified pectins were detected inside pollen tube cell vesicles and unesterified pectins were localized to the pollen tube wall. The in-vivo pollen tubes adhere to each other and can be separated by pectinase treatment. At the LM level, AGP localization occurred in the tube tip of both in-vivo- and in-vitro-grown pollen tubes and, in the case of one AGP probe, on the surface of the TTEs. Another AGP probe localized to every cell of the style except the surface of the TTE. At the TEM level, AGPs were mainly found on the plasma membrane and vesicle membranes of in-vivo-grown pollen tubes as well as on the TTE surface, with some localization to the adhesion zone between pollen tubes and style. (-d-Glc)₃ Yariv reagent bound to the in-vitro-grown pollen tube tip and significantly reduced the growth of both in-vitro- and in-vivo-grown pollen tubes. This led to abnormal expansion of the tube tip and random deposition of callose. These effects could be overcome by removal of (-d-Glc)₃ Yariv reagent which resulted in new tube tip growth zones emerging from the flanks of the arrested tube tip. The possible roles of pectins and AGPs in adhesion during pollination and pollen tube growth are discussed.Abbreviations AGP arabinogalactan-protein - ECM extracellular matrix - Glc glucose - MAbs monoclonal antibodies - LM light microscope - Man mannose - TEM transmission electron microscope - TTE transmitting tract epidermal cell The authors thank Michael Georgiady for assistance with the preparation of material for the TEM immunolocalization, Diana Dang for her help with the pectinase experiment, and Kathleen Eckard for assistance in all aspects of this study. The MAbs were the generous gifts of Dr. J.P. Knox. G.Y. Jauh thanks Dr. E.A. Nothnagel for assistance in making the Yariv reagent and for the gift of the control (-d-Man)₃ Yariv reagent. This work is in partial fulfilment of the dissertation requirements for a PhD degree in Botany and Plant Sciences for G.Y. Jauh at the University of California, Riverside. This work was supported by National Science Foundation grant 91-18554 and an R.E.U. grant to E.M.L.     

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.     

Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth

Vacuolar sorting receptors (VSRs) are type‐I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type‐IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans‐Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP‐VSR/GFP‐LIVSR is located throughout the pollen tubes, excepting the apical clear‐zone region, whereas GFP‐LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.     

Development and cellular organization ofPinus sylvesfris pollen tubes

The organization ofPinus sylvestris pollen tubes during growth was studied by video microscopy of living cells and by electron microscopy after freeze-fixation and freeze-substitution (FF-FS). Pollen germinated and the tubes grew slowly for a total period of about 7 days. Some of the grains formed two tubes, while 10–50% of the tubes ramified. These features are in accordance with development in vivo. The cytoplasmic hyaline cap at the tip disappeared during the 2nd or 3rd day of culture. Aggregates of starch grains progressively migrated from the grain into the tube and later into the branches. Vacuoles first appeared at day 2 and eventually filled large parts of the tube. The tube nucleus was located at variable distances from the tip. Some of the organelles showed linear movements in a mostly circulatory pattern, but the majority of the organelles showed brownian-like movements. Rhodamine-phalloidin-stained actin filaments had a gross axial orientation and were found throughout the tube including at the tip. The ultrastructure of pollen tubes was well preserved after FF-FS, but signs of shrinkage were visible. The secretory vesicles in growing tips were not organized in a vesicle cone, and coated pits had a low density with only local accumulations, which is in accordance with slow growth. The mitochondria contained small cristae and a darkly stained matrix and were located more towards the periphery of the tube, indicating low respiratory activity and low oxygen levels. The dictyosomes carried typical trans-Golgi networks, but some contained less than the normal number of cisternae. Other elements of the cytoplasm were irregularly spaced rough endoplasmic reticulum, many multivesicular bodies, lipid droplets and two types of vacuoles. The typical organization associated with tip growth in angiosperm pollen tubes, e.g.Nicotiana tabacum, was not present inP. sylvestris pollen tubes. The different morphology may relate to the growth rate and not to the type of growth.     

Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility

 Style squashes and stylar grafts were used to examine the growth of Nicotiana alata pollen tubes in self-compatible and self-incompatible styles. Compatible tubes typically showed a uniform layer of callose deposition in the walls and in small plugs spaced at regular intervals within the tube. Incompatible tubes were characterised by the variability of callose deposition in the walls and by larger, closer and more irregularly spaced plugs. There was no difference in the growth rate of compatible and incompatible tubes during growth through the stigma, but within the style most compatible tubes grew 20–25 mm day^-1 (maximum 30 mm day^–1), whereas incompatible tubes grew 1.0–1.5 mm day^-1 (maximum 5 mm day^–1). Many incompatible tubes continued to grow until flowers senesced, and only a small proportion died as a consequence of tip bursting. Grafting compatibly pollinated styles onto incompatible styles showed that the incompatible reaction could occur in pollen tubes between 2 and 50 mm long, and that inhibition of pollen tube growth occurred in both the upper and lower parts of the transmitting tract. Grafting incompatibly pollinated styles onto compatible styles showed that the incompatible reaction was fully reversible in at least a proportion of the pollen tubes. The findings are not consistent with the cytotoxic model of inhibition of self-pollen tubes in solanaceous plants, which assumes that the incompatible response results from the degradation of a finite amount of rRNA present in the pollen tube. However, if pollen tubes do in fact synthesise rRNA, the findings become consistent with this model. Received: 23 May 1996 / Revision accepted: 22 August 1996     

Movement of generative cell and vegetative nucleus in tobacco pollen tubes is dependent on microtubule cytoskeleton but independent of the synthesis of callose plugs

The role of microtubules (MTs) in vegetative nucleus (VN) and generative cell (GC) transport was investigated by comparing VN and GC distribution with callose plug formation in tobacco pollen grains germinated and grown for 12 h with the plant-specific anti-MT drug oryzalin. The VN-GC complex or VN alone was located close to the tube tip in 100% of controls, but in only 5% of oryzalin-treated tubes. Instead, in 38% of oryzalin tubes, the complex or VN occurred close to the last-formed callose plug; in 40% between or in the middle of plugs; and in 17%, in or near the grain. An aberrant microfilament (MF) cytoskeleton was revealed by expression of a green fluorescent protein-talin fusion protein in living oryzalin-treated tubes. The abnormal MF structures probably resulted from the absence of MTs and impaired - or were a consequence of - VN and GC movement into the tube tip. In oryzalin tubes with several callose plugs, the VN and GC could be in or near the grain, indicating that callose plug synthesis is not dependent on the movement of VN and GC into the tube. VN and GC movement and callose plug formation are apparently independent events, in which the transport of the VN-GC complex must precede callose plug synthesis. Maintenance of the correct developmental program requires an intact MT cytoskeleton, otherwise no fertile pollen tubes are formed.     

In vitro inhibition of incompatible pollen tubes in <Emphasis Type="Italic">Nicotiana alata</Emphasis> involves the uncoupling of the F-actin cytoskeleton and the endomembrane trafficking system

In the S-RNase-based self-incompatibility system, subcellular events occurring in the apical region of incompatible pollen tubes during the pollen rejection process are poorly understood. F-actin dynamics and endomembrane trafficking are crucial for polar growth, which is temporally and spatially controlled in the tip region of pollen tubes. Thus, we developed a simple in vitro assay to study the changes in the F-actin cytoskeleton and the endomembrane system at the apical region of incompatible pollen tubes in Nicotiana alata. Growth but not germination of pollen tubes of S _c10 -, S ₇₀ -, and S ₇₅ -haplotypes was selectively inhibited by style extracts carrying the same haplotypes. Pollen F-actin cytoskeleton and endomembrane system, visualized by fluorescent markers, were normal during the initial 60 min of pollen culture in the presence of compatible and incompatible style extracts. Additional culture resulted in complete growth arrest and critical alterations in the integrity of the F-actin cytoskeleton and the endomembrane system of incompatible pollen tubes. The F-actin ring and the V-shaped zone disappeared from the apical region, while distorted F-actin cables and progressive formation of membrane aggregates evolved in the subapical region and the shank. The vacuolar network of incompatible pollen tubes invaded the tip region, but vacuolar membrane integrity remained mostly unaffected. The polar growth machinery of incompatible pollen tubes was uncoupled, as evidenced by the severe disruption of colocalization between the F-actin cytoskeleton and the endomembrane compartments. A model of pollen rejection integrating the main subcellular events occurring in incompatible pollen is discussed.     

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.     

Circular F-actin bundles and a G-actin gradient in pollen and pollen tubes of Lilium davidii

The distribution of and relationship between F-actin and G-actin were investigated in pollen grains and pollen tubes of Lilium davidii Duch. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. Circular F-actin bundles were found to be the main form of microfilament cytoskeleton in pollen grains and pollen tubes. Consistent with cytoplasmic streaming in pollen tubes, there were no obvious F-actin bundles in the 10- to 20-microm tip region of long pollen tubes, only a few short F-actin fragments. Labeling with fluorescein isothiocyanate (FITC)-DNase I at first established the presence of a tip-focused gradient of intracellular G-actin concentration at the extreme apex of the tube, the concentration of G-actin being about twice as high in the 10- to 20-microm region of the tip as in other regions of the pollen tube. We also found that the distribution of G-actin was related negatively to that of the F-actin in pollen tubes of L. davidii. Caffeine treatment caused the G-actin tip-focused gradient to disappear, and F-actin to extend into the pollen tube tip. Based on these results, we speculate that the circular F-actin bundles may be the track for bidirectional cytoplasmic streaming in pollen tubes, and that in the pollen tube tip most of the F-actin is depolymerized into G-actin, leading to the absence of F-actin bundles in this region.     

Studies ofNajas pollen tubes. Fine structure and the dependence of chlorotetracycline fluorescence on external free ions

Summary The distribution of membrane-associated calcium was investigated in pollen grains and tubes of the underwater pollinated angiospermNajas marina L. using chlorotetracycline (CTC). Tubes grown in distilled water (pH 6) showed the highest fluorescence in a subapical region that tapered basally into a fluorescent strand centrally located in the tube and extending back towards the pollen grain. The apical cap had low fluorescence as did the cytoplasm surrounding the fluorescent strand, the tube base and the pollen grain. Tubes grown in different pond waters (pH 8) revealed no intracellular CTC fluorescence. Instead there was an external fluorescence forming a distinct layer around the whole tube, frequently enhanced in a subapical region to form an external collar.Modification of the patterns of fluorescence could be induced by manipulation pH of the growth media and content of specific ions. For example tubes grown in distilled water with 10^–3 M Mg²⁺ salts showed a similar CTC fluorescence as those grown in pond water. In contrast, Ca²⁺ enrichment had no visible influence on the patterns of fluorescence. The pattern of fluorescence displayed by tubes grown in distilled water, could be reproduced in pond water if the pH was artificially reduced to pH 6.Ultrastructurally, there was no detectable difference in the markedly polar distribution of organelles between pollen tubes grown in the various growth media. The secretory vesicles found in the pollen grain prior to germination become distributed throughout the pollen tube but are least concentrated in regions that show highest internal CTC fluorescence. These regions appear to have large amounts of endoplasmic reticulum and include mitochondria.These results are discussed in relation to the significance of calcium gradients for tip growth and limitations in the use of CTC.Abbreviations CTC chlorotetracycline - SV secretory vesicle - ER endoplasmic reticulum - PIXE proton induced X-ray emissions     

Cotton embryogenesis: The tissues of the stigma and style and their relation to the pollen tube

Summary The stigma of cotton (Gossypium hirsutum) is covered by unicellular hairs. The cytoplasm of these hairs degenerates before the stigma becomes receptive. The vacuole remains intact, but the hair cytoplasm becomes a mass of dark, amorphous material with only a few organelles still being visible. The rest of the stigma consists of thin-walled parenchyma cells with large vacuoles and large amounts of starch. The cells of the style are differentiated into a uniseriate epidermis, vascular tissue, a cortex of thin-walled, vacuolate parenchyma cells, and the transmitting tissue. This latter tissue occupies the center of the style and consists of thick-walled cells with few vacuoles. The cells are rich in starch, ribosomes, endoplasmic reticulum and dictyosomes. They also contain deposits of calcium salts in the form of druses. The pollen germinates on the stigmatic hairs, grows down the outside of the hair and between the cells of the stigma to the transmitting tissue of the style. There the tubes grow between the walls of the cells but do not enter the cells themselves. Some transmitting cells adjacent to the pollen tube degenerate after the tip of the pollen tube has grown past them. However, not all degenerate, and those that do show no fixed spatial relationship to one another. The cells which do degenerate follow a characteristic pattern of breakdown. No ultrastructural evidence was found for the secretion of hydrolytic enzymes by the pollen tube.