The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.Abbreviations AE-LPLC anion-exchange low-pressure liquid chromatography - AMPPNP 5-adenylylimidodiphosphate - PKH pollen kinesin homologue - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Exocytosis in non-plasmolyzed and plasmolyzed tobacco pollen tubes

Exocytosis occurring during deposition of secondary wall material was studied by freeze-fracturing ultrarapidly frozen non-plasmolyzed and plasmolyzed tobacco pollen tubes. The secondary wall of tobacco pollen tubes shows a random orientation of microfibrils. This was observed directly on fractures through the tube wall and indirectly as imprints of microfibrils on fracture faces of the plasma membrane of non-plasmolyzed tubes. About half of the plasmatic fracture faces from non-plasmolyzed and plasmolyzed pollen tubes carried hexagonal arrays of intramembraneous particles in between randomly distributed particles. Deposition of secondary wall material was often accompanied by an undulated plasma membrane and the presence of membrane-bound vesicles in invaginations of the plasma membrane, between the plasma membrane and secondary wall and-especially in plasmolyzed tubes-within the secondary wall of tube flanks and wall cap. The findings are discussed in connection with published schemes of membrane behaviour during exocytosis.Abbreviations EF extraplasmatic fracture face - IMP(s) intramembraneous particle(s) - PF plasmatic fracture face Extended version of a contribution (poster) presented at the 8th Int. Symp. on Sexual Reproduction in Seed Plants, Ferns and Mosses, Wageningen, The Netherlands, August 1984 Dedicated to Prof. Dr. H.F. Linskens (Nijmegen) on the occasion of his 65th birthday in 1986     

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)     

Ultrastructure of cell wall and plugs of tobacco pollen tubes after chemical extraction of polysaccharides

Tobacco pollen tubes grown in vitro and from pollinated tobacco styles were treated by chemical solvents to remove one or more of the following polysaccharides from the tube walls: pectin (ethylenediamine tetraacetic acid); hemicellulose (alkali); callose (alkali; potassium hypochlorite); cellulose (cuprammonium); and all polysaccharides with exception of cellulose (H₂O₂/glacial acetic acid). Both the inner tube wall, which we had regarded as the secondary wall, and the plugs contained, in addition to callose, microfibrils of cellulose and non-cellulosic microfibrils that had pectin-like properties. When using the expressions callosic or callose layer and callose plugs in reference to pollen tubes, one should realize that they do not imply the exclusive presence of callose in the inner tube wall layer and its localized thickenings.Extended version of a contribution (poster) presented at the International Symposium Advances in Plant Cytoembryology in Lublin, Poland, in June 1980 Dedicated to Professor J. Straub (Köln-Vogelsang) on his 70th birthday in 1981     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

Organization of the cytoskeleton in pollen tubes ofPyrus communis: a study employing conventional and freeze-substitution electron microscopy,immunofluorescence, and rhodamine-phalloidin

Summary The structure and organization of the cytoskeleton in the vegetative cell of germinated pollen grains and pollen tubes ofPyrus communis was examined at the ultrastructural level via chemical fixation and freeze substitution, and at the light microscopic level with the aid of immunofluorescence of tubulin and rhodamine-phalloidin.Results indicate that cortical microtubules and microfilaments, together with the plasma membrane, form a structurally integrated cytoskeletal complex. Axially aligned microtubules are present in cortical and cytoplasmic regions of the pollen grain portion of the cell and the distal region of the pollen tube portion. Cytoplasmic bundles of microfilaments are found in association with elements of endoplasmic reticulum and vacuoles. Axially aligned microfilaments are also found in this region, associated with and independent of the microtubules. Microtubules are lacking in the subapical region where short, axially aligned microfilaments are found in the cell cortex. In the apical region, which also lacks microtubules, a 3-dimensional network of short microfilaments occurs. Microfilaments, but not microtubules, appear to be associated with the vegetative nucleus.     

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     

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     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Developmentally regulated antigen associated with calcium crystals in tobacco anthers

We have found and characterized an antigen associated with crystal-containing cells in the stomium and connective tissue of the anthers of Nicotiana tabacum L. (tobacco). The antigen, defined by the monoclonal antibody NtF-8B1, localizes to subcellular regions surrounding the crystals. At the light-microscope level, the antigen is detectable just after the first appearance of crystals in the connective tissue of the anther, and at approximately the same time as the appearance of crystals in the stomium. The antigen is not detectable on a Western blot, and gave inconclusive results on a test of periodate sensitivity. It is not the crystals themselves, nor is the presence of the crystals required for antibody recognition. The antigen is sensitive to heat and protease treatment, indicating that it is a protein. The antigen is not tightly membrane-bound, in spite of its localization closely surrounding the crystals. Chemical tests indicate that the druse crystals in the stomium are calcium oxalate.Abbreviations ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate This research was supported by a National Science Foundation postdoctoral fellowship to B.L.H., by National Science Foundation grants DMB-87-15799 and to W.E.F. BSR-88-18035, and by U.S. Department of Agriculture grant GAM-89-01056. The authors thank Phillip T. Evans (Louisiana State University, Baton Rouge, USA), Wilma L. Lingle, Harry T. Horner, Jr. (Iowa State University), and A. Jack Fowler, Jr., for advice and helpful discussions.     

Plasmatubules in the pollen tubes of Nicotiana sylvestris

Ultrastructural studies of the pollen tubes of Nicotiana sylvestris grown in the pistil revealed an extensive development of plasmatubules formed by evaginations of the plasma membrane. The plasmatubules occurred as twisted tubular structures in the periplasmic space along the tube wall and, in cross section, exhibited circular profiles with an outer diameter of 28±4 nm. They were also seen in deep, pocket-like invaginations of the plasma membrane and in this case the profiles had an outer diameter of 34±8 nm. In the pocket-like invaginations they were partially branched and often closely packed to form groups with obvious patterns. The enlargement of the plasma-membrane area resulting from plasmatubules formed along the tube wall was about six-to tenfold. Pollen tubes grown in vitro exhibited poorly developed plasmatubules. It is suggested that the large extension of the plasma membrane could enhance the uptake of nutrients, and thus might be responsible for the comparatively fast growth of pollen tubes in the pistil. Moreover, it is also assumed that the turnover rate of the Golgi apparatus must be higher in pollen tubes growing in vivo than in vitro, in order to provide a sufficient amount of membrane for the formation of the plasma membrane with its tubular modifications.     

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.     

Silencing of the tobacco pollen pectin methylesterase NtPPME1 results in retarded in vivo pollen tube growth

Sperm delivery in flowering plants requires extensive pollen tube growth through the female sporophytic tissues of the pistil. The apical cell wall emerges as a central player in the control of pollen tube growth, since it provides strength to withstand the internal turgor pressure, while imparting sufficient plasticity to allow cell wall extension through the incorporation of new membrane and wall material. Within this scenario, pectin methylesterases (PMEs; EC 3.1.1.11) emerge as crucial regulators in determining the mechanical properties of pectins, the major component of the apical pollen tube wall. We previously identified NtPPME1, a pollen specific PME from Nicotiana tabacum. Here we show that silencing of NtPPME1 results in a mild but significant decrease of in vivo pollen tube growth while the overall PME activity in pollen is not significantly affected. Although the precise mechanisms responsible for the observed phenotype are not known, it seems likely that the cell must maintain a closely regulated level of PME activity in order to maintain the equilibrium between strength and plasticity in the apical cell wall. A relatively minor disturbance of this equilibrium, as caused by NtPPME1 silencing, compromises pollen tube growth.     

Organization of actin filaments in regenerating and outgrowing subprotoplasts from pollen tubes ofNicotiana tabacum L.

The dynamics of actin-filament organization in pollen-tube subprotoplasts ofNicotiana tabacum L. cv. Samsun during regeneration and outgrowth was examined using phalloidin probes and a non-fixation method. A succession of actin arrays was examined during subprotoplast regeneration that strongly resembled the actin dynamics described for developing microspores by Van Lammeren et al. (1989, Planta178, 531–539) and activated pollen by Tiwari and Polito (1988, Protoplasma147, 5–15). At the end of the succession the actin filaments often became extended between two opposite polar foci. The ordering of the cortical actin filaments reflected a polarity in the subprotoplasts which determined the plane of outgrowth. The site of outgrowth was often marked by a ring of actin filaments. As growth proceeded and tube-like structures were formed, the arrangement of cortical actin filaments was found to be transverse to the elongation axis. Since the patterns of actin distribution were identical in both caryoplasts and cytoplasts, it was concluded that the pollen-tube cytoplasm has the intrinsic capacity of reorganizing actin filaments and imposing polarity on the spherical subprotoplasts.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Pentachlorophenol affects mitochondria and induces formation of golgi apparatus-endoplasmic reticulum hybrids in tobacco pollen tubes

Summary The toxic effect of pentachlorophenol (PCP) on the growth and ultrastructure of tobacco pollen tubes was tested using a semivivo technique of tube culture. In this technique the pollen tubes were allowed to grow in the pistilin situ for 24 hr before they protruded from the cut end of the style and came into contact with the medium containing PCP. The inhibitory effect of different PCP concentrations was determined by measuring the length of tube bundles. The intracellular action of PCP was analysed by electron microscopy. This biocide caused four obvious alterations in the pollen tube ultrastructure: (1) swelling of the mitochondrial saccules; (2) enlargement of the dictyosomes by the increase of the cisternal diameter and the number of cisternae per stack; (3) formation of cup-shaped Golgi apparatus-endoplasmic reticulum hybrid structures (GER hybrids) showing continuities of ER and Golgi cisternae; (4) formation of stacked and/or concentric arrangements of rough ER cisternae. It is suggested that swelling of saccules was directly due to the uncoupling of oxidative phosphorylation whereas the changes of the endomembrane system were caused by energy depletion due to the inhibition of ATP synthesis. These changes are consistant with dynamic concepts of dictyosome and ER function when membrane formation exceeds membrane use in the production of secretory and transition vesicles. Thus, the enlargement of the dictyosomes and the formation of GER hybrids are thought to result from inhibition of budding of vesicles from the Golgi apparatus or from both the ER and the Golgi apparatus, respectively.     

Composition of the cell walls of Nicotiana alata Link et Otto pollen tubes

Cell walls isolated from pollen of Nicotiana alata germinated in vitro contain glucose and arabinose as the predominant monosaccharides. Methylation analysis and cytochemical studies are consistent with the major polysaccharides being a (13)--D-glucan (callose) and an arabinan together with small amounts of cellulose. The cell walls contain 2.8% uronic acids. Alcian blue stains the pollen-tube walls intensely at the tip, indicating that acidic polysaccharides are concentrated in the tip. Synthetic aniline-blue fluorochrome is specific primarily for (13)--D-glucans and stains the pollen-tube walls, except at the tip. Protein (1.5%), containing hydroxyproline (2.4%), is present in the cell wall.     

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