Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

In vitro germination and growth of lily pollen tubes is affected by calcium inhibitor with reference to calcium distribution

The calcium inhibitors A23187, EGTA and La³⁺ inhibit pollen grain germination and growth of pollen tubes of Lilium davidii var. unicolor at different concentrations. Treatment with 10⁻⁴ or 10⁻⁵ M ionophores A23187 reduced germination rate and resulted in distortion of pollen tube. Addition of 2 or 10 mM of the chelator EGTA disturbed the direction of pollen tube growth and extended the diameter of pollen tube as observed by light and confocal microscopy. The Ca²⁺-channel blocker lanthanum chloride (La³⁺) restrained germination or markedly caused transformation of pollen tube. Furthermore, all treatments led to disappearance of any calcium gradient. Calcium distribution in pollen grain and pollen tube was altered as shown by confocal microscopy for each treatment. This indicates that the inhibitors influence pollen development by affecting the calcium gradient which may play a critical role in germination and tube growth. Fourier transform infrared (FTIR) spectra indicated slight increases in contents of amide I and a substantial decrease in the content of aliphatic esters and saturated esters in treated pollen tubes compared with normal pollen tubes. The FTIR analysis confirmed that EGTA and La³⁺ weakened the accumulation of ester in pollen tubes, which may be associated with an increased content of amide I.     

Cytochalasin B Treatment of Apple (<Emphasis Type="Italic">Malus pumila</Emphasis> Mill.) Pollen Tubes Alters the Cytoplasmic Calcium Gradient and Causes Major Changes in the Cell Wall Components

It is well established that the actin cytoskeleton is absolutely essential to pollen germination and tube growth. In this study we investigated the effects of cytochalasin B (CB), which affects actin polymerization by binding to the barbed end of actin filaments, on apple (Malus pumila Mill.) pollen tube growth. Results showed that CB altered the morphology of pollen tubes, which had a larger diameter than control tubes beside inhibiting pollen germination and tube growth. Meantime CB also caused an abnormal distribution of actin filaments in the shank of the treated pollen tubes. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium ([Ca²⁺]c) in the pollen tube tip was abolished by exposure to CB, which induced a much stronger signal in the cytoplasm. Cellulose and callose distribution in the tube apex changed due to the CB treatment. Immunolabeling with different pectin and arabinogalactan protein (AGP) antibodies illustrated that CB induced an accumulation of pectins and AGPs in the tube cytoplasm and apex wall. The above results were further supported by Fourier-transform infrared (FTIR) analysis. The results suggest the disruption of actin can result in abnormal growth by disturbing the [Ca²⁺]c gradient and the distribution of cell wall components at the pollen tube apex.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.     

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     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

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.     

Development of membrane- and calcium-gradients during pollen germination of Lilium longiflorum

Chlorotetracyclin (10^-4M) has been used to observe the distribution of membrane-associated calcium during pollen germination of Lilium longiflorum. For comparison, the general membrane distribution has been determined with 4·10^-5 M fluorescamine. The pollen grains show a calcium gradient with either weak or strong chlorotetracycline-fluorescence intensity, but always increasing toward the germination colpus. This gradient intensifies during germination, reaching a maximum before the pollen tube emerges. The typical tip-to-base calcium gradient of the tube does not change during growth. Independent of the developmental stage, the pollen grains show a flat fluorescamine-fluorescence gradient with the highest intensity in one half of the grain. Pollen tubes reveal a tip-to-base membrane gradient, independent of their length. As an additional marker for membrane distribution, the distribution of phosphorus, measured by proton-induced X-ray emission in chemically fixed tubes, has been used. A tip-to-base phosphorus gradient, distinct from the calcium gradient measured with the same method, was detected.Abbreviation CTC chlorotetracycline     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Wide hybridization: pollination of Zea mays L. by Sorghum bicolor (L.) Moench

Summary Foreign pollen tubes in the stigma of Zea mays can be prevented from reaching the ovary cavity by the unusual length of the pollen tube pathway. A simple and rapid procedure is described for overcoming this difficulty by pollinating the basal parts of the stigmas without removing the ensheathing bracts (husks). The method maintains high humidity in the vicinity of the ovaries, and by conserving photosynthetic tissues probably also ensures a more normal O₂ /CO₂ balance in the neighbourhood of the stigmas than do bagging procedures. It is shown that Sorghum pollen tubes readily reach the ovary after pollination by the method. Their presence induces some of the characteristic post-pollination effects caused by Zea pollen tubes, but they frequently also stimulate premature enlargement of the nucellus and lysis of nucellar cells. Although Sorghum tubes have been traced across the inner ovary wall, they have not been seen to enter the micropyle, and hybrid embryos have not yet been obtained.     

Inhibition of pollen-tube growth and protein secretion by the monovalent ionophore monensin

Summary Apple pollen tubes grown in vitro in the presence of up to 1 M monensin, which is known to slow down or stop vesicular traffic originating from the Golgi complex, were severely inhibited by the carboxylic ionophore. Monensin at concentrations as low as 5 nM significantly decreased the secretory release of protein into the extracellular space. During 2 h of germination in the presence of monensin, ³H-leucine incorporation into the membrane versus the soluble fraction increased with respect to the controls. These data seem to be consistent with an effective action of monensin on the secretory routes which, in pollen tubes, deliver products from the dictyosomes to the extending tip. The importance of monovalent cation homeostasis in the functioning of secretory paths, also in unique cells such as pollen tubes, emerges clearly from the present data.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca<Superscript>2+</Superscript> concentration

Cytosolic free calcium ([Ca²⁺]_cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca²⁺]_cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K⁺] and [Ca²⁺]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca²⁺]_cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.     

Different effects of inorganic and triethyl lead on growth and ultrastructure of lily pollen tubes

Summary Pollen tubes ofLilium longiflorum growingin vitro were treated for 1 h with inorganic lead (Pb) and with triethyl lead (TriEL) and studied by light and electron microscopy. Pb was considerably more toxic in relation to inhibition of pollen tube growth (EC₅₀=6 M Pb) than was TriEL (EC₅₀=60 M TriEL). On the other hand, at almost the entire concentration range tested (25-500 M) TriEL caused aberrant tubes and tube swellings. Pb did not cause tube swellings, even at highly growth-impairing concentrations. Pb (60 M) predominantly affected the ultrastructure of the growing cell walls without impairing the distribution of the cell organelles in the tube tips. In contrast, 50 and 100 M TriEL did not visibly influence cell wall ultrastructure but it severely damaged dictyosomes; 100 M TriEL also disturbed the original order of cell organelles in the tube tips. Cortical microtubules were selectively and completely destructed by TriEL at concentrations (50 M) where no effect on polar organization of the tube tips occurred but they remained unimpaired by 60 M Pb, indicating selective and effective interaction of TriEL with these cell organelles.Abbreviations EC⁵⁰ effective lead concentration causing 50% inhibition of pollen tube growth - MTs microtubules - Pb inorganic lead - TriAL trialkyl lead - TriEL triethyl lead     

Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris

Summary In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maximum length of 25 m. With a solution based on Monnier's medium 47% germination and 160-mlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/l calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/l, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/l each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/l (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length.     

Relationship between the generative cell and vegetative nucleus in pollen tubes of Nicotiana tabacum

Summary Fluorescence microscopy was used to visualize microtubules (Mts) and chromatin in an effort to further clarify the relationship between the generative cell (GC) and vegetative nucleus (VN) in pollen tubes of tobacco. Prominent Mt bundles are present in one or more GC extensions that can be finger-like or lamellar in form. While the VN is positioned distal to the GC in most cases, it can also straddle the cell or lie proximal to it. In all cases, however, extensions embrace, penetrate or clasp the VN. GC Mts are reorganized during the formation of the mitotic apparatus, and cell extensions are fully or partially withdrawn. By telophase in many pollen tubes, the VN shifts to a more proximal position and appears to adhere to the region of the GC containing the phragmoplast. Application of oryzalin leads to the disorganization of Mts, changes in cell shape, including the loss or alteration of cell extensions, and separation of the GC and VN in some cases. However, the position and polarity of the VN is maintained in most pollen tubes. The results indicate that GC Mts and cell extensions play a role in the association with the VN. However, the relationship appears to be controlled by other factors as well. Attention should now be directed at potential interactions involving the VN envelope, vegetative plasma membrane, GC plasma membrane and extracellular matrix.Abbreviations GC Generative cell - MGU male germ unit - Mt microtubule - VN vegetative nucleus     

Regulation der Nucleinsäuren-Synthese durch Polyamine in keimendem Pollen vonPetunia

Summary Putrescine, spermine, spermidine, and agmatine in concentrations between 5–15 g/ml inhibit pollen germination. Whereas spermine reduces pollen tube length, putrecine and agmatine do not affect pollen tube growth. Spermidine effects a small increase (about 5%) of pollen tube elongation. Spermine and spermidine can be found in pollen. Addition of spermine (7 or 10 g/ml) depresses protein synthesis, whilst spermidine does not affect protein synthesis. On the basis of uridine-5-T incorporation it could be shown that both spermine and spermidine increase RNA synthesis. On tho basis of thymidine-T incorporation in the first hpurs of germination it seems that DNA synthesis is also stimulated by spermine and spermidine present in the medium. A net increase of nucleic acids was found when spermidine was added to the germination substrate.These results are interpreted as suggesting that, in the pollen tubes investigated, polyamine concentration may be a factor in the regulation of nucleic acid synthesis, resulting in a prolonged synthesis of specific proteins and in this way influencing growth and the developmental pattern of pollen tubes.     

A cytochemical study on the role of ATPases during pollen germination in Agapanthus umbelatus L'Her.

Summary Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca²⁺-ATPases were mainly localized in mitochondria and ER membranes, while Mg²⁺-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K⁺-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.The results are discussed in terms of the ultrastructural organization of the PG and PT, especially those correlated with (1) formation and maintenance of ionic gradients throughout the PT, (2) polarized growth and (3) hydrodynamics of PT elongation.Abbreviations PT Pollen tube - PG pollen grain - PTW pollentube wall - PGW pollen-grain wall - ER endoplasmic reticulum - NEM N-ethylmaleimide     

Ionophore A 23187 stops tip growth,but not cytoplasmic streaming,in pollen tubes ofLilium longiflorum

Summary The effects of the cationophore A 23187 on growing pollen tubes ofLilium longiflorum and on pollen germination were testedin vitro, and measured light microscopically. The ionophore is a very potent inhibitor of pollen tube growth: ionophore contentrations down to 10^–7 M stop tip growth. Cytoplasmic streaming is less sensitive: Only with added external Ca²⁺ and higher concentrations of the ionophore the cytoplasmic streaming is stopped. Pollen germination is less sensitive to ionophore than pollen tube growth at later stages. The ionophore inhibition is partially reversible in a medium containing no added external Ca²⁺, but is not reversible in a Ca²⁺-enriched medium. EDTA addition to the medium prevents pollen germination and growth totally. It is hypothesized that the pollen ofLilium longiflorum needs Ca²⁺ to sustain oriented exocytosis at the pollen tube tip. The ionophore A 23187 seems to interfere with the electrical pulse/Ca²⁺-orientation mechanism of exocytosis by equilibration of the Ca²⁺-gradient.