Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10^-5 M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.Abbreviations CTC chlorotetracycline - DMSO dimethylsulfoxide     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

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.     

POLLEN TUBE GROWTH AND INCOMPATIBILITY FOLLOWING INTRA- AND INTER-SPECIFIC POLLINATIONS IN LILIUM LONGIFLORUM

The growth of pollen tubes of a sampling of Lilium species in styles of L. longiflorum incubated at 24 C for 48 hr after pollination indicated two types of interspecific incompatibility. Pollen tubes of two self-compatible species of section Leucolirion, the section including L. longiflorum, stopped growth abruptly upon reaching the stylar canal, were of abnormal morphology, and were incapable of continued growth with longer incubation. Pollen tubes of self-compatible or self-incompatible species of sections Sinomartagon, Pseudolirium, Liriotypus, and Daurolirion approached but did not exceed the length of intraspecific incompatible pollen tubes in styles of L. longiflorum. Pollen tube morphology was normal and tubes were capable of continued growth with additional incubation. Unilateral interspecific incompatibility occurred in reciprocal crosses between self-incompatible L. longiflorum and self-compatible L. regale and L. formosanum, but exceptions occurred in Aurelian hybrids. Incubation of interspecifically pollinated L. longiflorum styles at 39 C, which removes the self-incompatibility reaction, had no effect on interspecific incompatibility.     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style     

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     

Inhibition of Intracellular Pectin Transport in Pollen Tubes by Monensin,Brefeldin A and Cytochalasin D*

Specific inhibitors of the secretory pathway represent important tools for investigation of cell wall synthesis and tip growth in pollen tubes. Brefeldin A completely inhibits germination of Nicotiana tabacum pollen tubes at 2.2 μM. Ultrastructural investigation of pollen tube cytoplasm showed that brefeldin A caused the appearance of reticular structures and “brefeldin A compartments” containing unesterified pectins. Monensin caused inhibition of pollen tube germination at 80 nM. The drug induced swelling of the Golgi cisternae, many of which contained methyl-esterified pectins. Cytochalasin D was effective at 1 μg/ml, the inhibition of germination being fully reversible. Application of the drug caused accumulation of secretory vesicles containing methyl-esterified pectin around the dictyosomes. In contrast to brefeldin A and monensin, cytochalasin D caused a slowdown of cytoplasmic streaming. Monensin, but not the other drugs, caused a considerable decrease in pollen tube diameter. The characterization and quantification of the effects of the drugs on pollen tubes represents a necessary prerequisite for their application in physiological studies.     

Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein-NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein-NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.     

Visualization of the Ca2+-gradient in growing pollen tubes ofLilium longiflorum with chlorotetracycline fluorescence

Summary Epifluorescence microscopy with chlorotetracycline (CTC) fluorescence was used to visualize the Ca²⁺ distribution in germinating pollen grains and growing pollen tubes ofLilium longiflorum. The intensity of the fluorescence shows a gradient with the highest fluorescence at the growing tip. Added external Ca²⁺ influences the intensity of the gradient in germinating grains. Ionophore A 23187 treated pollen tubes do not show the fluorescence gradient with CTC. These results are interpreted as evidence for the role of a Ca²⁺ gradient in pollen tube tip growth.     

Myosin XI-B is involved in the transport of vesicles and organelles in pollen tubes of Arabidopsis thaliana

The movement of organelles and vesicles in pollen tubes depends on F-actin. However, the molecular mechanism through which plant myosin XI drives the movement of organelles is still controversial, and the relationship between myosin XI and vesicle movement in pollen tubes is also unclear. In this study, we found that the siliques of the myosin xi-b/e mutant were obviously shorter than those of the wild-type (WT) and that the seed set of the mutant was severely deficient. The pollen tube growth of myosin xi-b/e was significantly inhibited both in vitro and in vivo. Fluorescence recovery after photobleaching showed that the velocity of vesicle movement in the pollen tube tip of the myosin xi-b/e mutant was lower than that of the WT. It was also found that peroxisome movement was significantly inhibited in the pollen tubes of the myosin xi-b/e mutant, while the velocities of the Golgi stack and mitochondrial movement decreased relatively less in the pollen tubes of the mutant. The endoplasmic reticulum streaming in the pollen tube shanks was not significantly different between the WT and the myosin xi-b/e mutant. In addition, we found that myosin XI-B-GFP colocalized obviously with vesicles and peroxisomes in the pollen tubes of Arabidopsis. Taken together, these results indicate that myosin XI-B may bind mainly to vesicles and peroxisomes, and drive their movement in pollen tubes. These results also suggest that the mechanism by which myosin XI drives organelle movement in plant cells may be evolutionarily conserved compared with other eukaryotic cells.     

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

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

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.     

Pollen tubes of flavonol-deficient Petunia show striking alterations in wall structure leading to tube disruption

Despite the vital role that flavonols play in fertilization and pollen tube growth of a number of species such as petunia and maize, their function is still unclear. Pollen tubes of the flavonol-deficient transformant T17.02 of Petunia hybrida L. are able to germinate and start growing in vitro, but eventually disrupt at the tip approximately 2 h after germination. In order to establish the possible role of flavonols in this process, wild-type and flavonol-deficient pollen tubes were subjected to cytological and ultrastructural analyses and screened for differences. The results showed that before disruption of the flavonol-deficient pollen tubes, the structure of the primary wall at the tip dramatically changed from layered to granular. Secretory vesicles at the tip still fused with the wall but lost their capacity to melt into the wall and to form layers. Instead they remained as dark, electron-dense granular structures surrounded by an electron-translucent matrix. Apparently the matrix is not able to sustain the wall's coherence and as a consequence the tube disrupts. No other remarkable cytological or ultrastructural differences between the transformant and the wild-type pollen tubes could be found before tip disruption. Even a morphometric analysis of abundance and distribution of endoplasmic reticulum, dictyosomes and mitochondria did not reveal any significant difference. However, for the first time, obvious morphological differences were observed in the wall of the flavonol-deficient pollen tubes. We conclude that flavonols act on precursors of the pollen tube wall of petunia and interfere with a cross-linking system in the wall, possibly via extensins. Received: 23 February 1998 / Accepted: 13 August 1998     

Demonstration of polar zinc distribution in pollen tubes ofLilium longiflorum with the Heidelberg proton microprobe

Summary The zinc distribution in pollen tubes ofLilium longiflorum was analysed by proton-induced X-ray emission (PIXE) with the Heidelberg proton microprobe. A very high zinc concentration was measured in the pollen tube tip region.On leave from Shanghai Institute of Nuclear Research, Academia Sinica, Shanghai, China     

Immunolocalization and possible roles of pectins during pollen growth and callose plug formation in angiosperms

To elucidate the possible roles of pectins during the growth of angiosperm pollen, we studied the distribution and changes in the properties of pectin in the pollen grains and tubes of Camellia japonica, Lilium longiflorum, and five other species at different growth stages by immunoelectron microscopy with monoclonal antibodies JIM5, against de-esterified pectin, and JIM7, against esterified pectin. We also studied the localization of arabinogalactan proteins, which are regarded as pectin-binding proteins, with monoclonal antibodies JIM13 and LM2, against arabinogalactan proteins. Similar results were obtained for all species: JIM5 labeled the intine and part of the callose layer in germinated pollen grains, and labeled the outer layer of the tube wall, the Golgi vesicles, and the callose plug in the pollen germinated in vitro, but did not label any part of immature pollen grains. In contrast, JIM7 labeled the intine of both immature and mature pollen grains, labeled the Golgi vesicles around the Golgi bodies, and strongly labeled the outer layer of the cell wall and the Golgi vesicles in the tube tip region. On the other hand, the distribution of arabinogalactan proteins detected with JIM13 was different for each species, and does not suggest a close relationship between pectin and arabinogalactan proteins. LM2 scarcely reacted with the specimens. We discuss the contribution of pectins to tube wall formation and fertilization and deduce a mechanism of callose plug formation.     

Properties and partial protein sequence of plant annexins

We have examined the characteristics of Ca²⁺-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23. Partial sequence analysis reveals that peptides from p35 and p33 have identity to members of the annexin family of animal proteins and to annexins from tomato. Interestingly, multiple sequence alignments reveal that the domain responsible for Ca²⁺ binding in animal annexins is not conserved in these plant peptide sequences. Although p33 and p35 share the annexin characteristic of binding to membrane lipid, unlike annexins II and VI they do not associate with detergent-insoluble cytoskeletal proteins or with F-actin from either plants or animals. Immunoblotting with antiserum raised to p33/p35 from maize reveals that cross-reactive polypeptides of 33 to 35 kilodaltons are also present in protein extracts from pollen tubes of L. longiflorum. Immunolocalization at the light microscope level suggests that these proteins are predominantly confined to the nongranular zone at the tube tip, a region rich in secretory vesicles. Our hypothesis that plant annexins mediate exocytotic events is supported by the finding that p23, p33, and p35 bind to these secretory vesicles in a Ca²⁺-dependent manner.     

Location of cellulose and callose in pollen tubes and grains of Nicotiana tabacum

The distribution of cellulose and callose in the walls of pollen tubes and grains of Nicotiana tabacum L. was examined by electron microscopy using gold-labelled cellobiohydrolase for cellulose and a (1,3)-β-D-glucan-specific monoclonal antibody for callose. These probes provided the first direct evidence that cellulose co-locates with callose in the inner, electron-lucent layer of the pollen-tube wall, while both polymers are absent from the outer, fibrillar layer. Neither cellulose nor callose are present in the wall at the pollen-tube tip or in cytoplasmic vesicles. Cellulose is first detected approximately 5–15 μm behind the growing tube tip, just before a visible inner wall layer commences, whereas callose is first observed in the inner wall layer approximately 30 μm behind the tip. Callose was present throughout transverse plugs, whereas cellulose was most abundant towards the outer regions of these plugs. This same distribution of cellulose and callose was also observed in pollen-tube walls of N. alata Link et Otto, Brassica campestris L. and Lilium longiflorum Thunb. In pollen grains of N. tabacum, cellulose is present in the intine layer of the wall throughout germination, but no callose is present. Callose appears in grains by 4 h after germination, increasing in amount over at least the first 18 h, and is located at the interface between the intine and the plasma membrane. This differential distribution of cellulose and callose in both pollen tubes and grains has implications for the nature of the β-glucan biosynthetic machinery. Received: 20 February 1988 / Accepted: 25 March 1998     

SEC1A and SEC6 synergistically regulate pollen tube polar growth

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles. The exocyst and Sec1/Munc18 (SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through co-immunoprecipitation-mass spectrometry screening, protein interaction analysis and in vivo microscopy, we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.     

The effect of cycloheximide and 6-methylpurine on in vivo compatible and incompatible pollen tube growth in Lilium longiflorum

Summary Cycloheximide, a potent inhibitor of protein synthesis, placed in styles of Lilium longiflorum at 10^–4 M in stigmatic exudate before, 6, or 12 hr after compatible or incompatible pollination retarded all pollen tube growth. An inhibitor of RNA synthesis, 6-methylpurine, placed in the style at 10^–4 M in stigmatic exudate before, 6, or 12 hr after pollination restricted compatible pollen tube growth to lengths not significantly different thanincompatiblepollen tubes in treated or nontreated styles. While pollen tube growth in the style of L. longiflorum appears to require protein synthesis, only compatible pollen tube growth requires RNA synthesis. Stigmatic exudate proved to be an excellent carrier of exogenous substances into the style of L. longiflorum. Paper number 7047 of the Scientific Journal Series Minn. Agr. Exp. Sta. Research was supported in part by funds provided by the Graduate School, University of Minnesota.The authors wish to thank the United Bulb Co., Mount Clemens, Mich. for lily bulbs and L. H. Fuchigami and L. V. Gusta, Dept. of Hort. Sci., Univ. of Minn. for advice on use of the inhibitors. Mr. Drewlow is a National Science Foundation predoctoral trainee.