Confocal Microscopy Observations on Actin Cytoskeleton in the Pollen and Pollen Protoplast of Narcissus

Actin cytoskeleton was localized in the pollen and pollen protoplast of Narcissus cyclamineus using fluorescence labelled phalloidin andconfocal microscopy. In the hydrated pollen (before germination) actin filamem bundles were arranged in a parallel array and at right angles to the long axis of the pollen grain in the cortex. But at the germination pore region(or fur row) the actin filament bundles formed a reticulate network. In the centre of the grain there was also an actin filament network which was more open and had less bundles associated with it than the network underneath the furrow. When the pollen grain started to produce pollen tube, most(if not all) of the actin filament bundles in the pollen grain rearranged into a parallel array pointing towards the tube. The bundles in the array later elongated and extended into the pollen tube. In the pollen protoplast a very tightly-packed actin bundle network was present. Numerous branches and jonts of actin filament bundles could be seen in the network. If the protoplasts were fixed before staining, the bundles aggregated and the branches and joints became less obvious indicating that fixation had affected the nature and arrangement of the actin filament bundles. If the pollen protoplasts were bursted (using the osmotic shock technique) or extracted (using Triton X-100), fragments of actin filament bundles could still be found associated with the membrane ghost indicating that some of the actin filament bundles in the cortex were tightly attached to the membrane. Using a double staining technique, actin filaments and microtubules were co-localized in the pollen protoplast. The co-alignment of some of the actin filament bundles with the microtubule bundles suggested that the actin cytoskeleton and the microtubule cytoskeleton were not distributed at random but in a well organized and orchestrated manner [possibly under the control of a yet undiscovered structure(s). The actin filament cytoskeleton in the generative cells failed to stain either in pollen or pollen tube, but they became stained in the pollen protoplast. The actin cytoskeleton in the generative cell appeared as a loosely organized network made up of short and long actin filament bundles.     

Isolation of Pollen Protoplasts in Nicotiana tabacum

A new method for isolation of quantities of mature pollen protoplasts in Nicotiana tabacum has been established. The first step was to germinate mature pollen in Brewbaker and Kwack medium containing 20% sucrose. When most of the pollen grains had just germinated short pollen tubes, they were transferred to an enzymatic solution for the second step. The enzymatic solution contained 1% pectinase, 1% cellulase, 0.5% potassium dextran sulfate, 1 mol/L mannitol, 0.4 mol/L sorbitol in Dx medium with or without 15% Ficoll. The enzymes firstly degraded the pollen tube wall and then the intine. As a result, intact pollen protoplasts were released with the isolation rate up to 50%-70%. Factors affecting pollen protoplast isolation during the germination and maceration of pollen grains were studied. The suceees depended on two key points:pollen germination duration and osmotieum concentration. The optimal germination duration was 30 rain at 30℃. When it was too long, long pollen tubes formed and subsequently, large number of subprotoplasts instead of whole protoplasts were yielded, as the case reported by previous investigators. The optimal concentration of mannitol and sorbitol in enzyme solution was as high as 1.4 mol/L in total. Lowering of the osmoticum concentration resulted in decrease of percentage of pollen protoplasts.     

Confocal Microscopic Observations on Actin Filament Distribution in Lily Pollen Protoplasts

Actin filaments (F-actin) were localized in the isolated pollen protoplasts of lily using TRITC-phalloidin probe and confocal microscopy. Two kinds of pollen protoplasts were examined: one from pollen grains of non-dehiscent anthers(referred to as ‘nearly mature’ pollen); and the other from pollen grains of just dehiscent anthers(referred to as ‘just mature’ pollen). In the cytoplasm of the pollen protoplasts of the ‘nearly mature’ pollen there was a very well organized actin network made up of thick actin bundles. Two types of bundle connections were seen in the network; namely ‘branch’ connections and 'junction' connections. The ‘branch’ connection (or branching points) was formed due to branching or merging of bundies. The ‘junction’ connection (or 'junction' point) had two or more bundles associated with it. Some of the ‘junction’ points might be actin filament organization: centres. The generative cell in iht pollen protoplasts of the ‘nearly mature’ pollen also contained an actin network. But this network was structurally quite loose and the pundles made up the network were short and thick. In the cytoplasm of the pollen protoplasts of the ‘just mature’ pollen the actin net work was more densely packed. The bundles made up the network were also thinner. The actin network in the generative cell was, however, less densely packed. If the pollen protoplasts from both the ‘nearly mature’ and the 'just mature' pollen grains were transferred from a B5 medium into a Brewbaker and Kwack medium supplemented with sucrose, protoplasts rapidly (i.e. within 2 to 3 hours) developed vacuoles and transvacuolar strand. In these va cuolated protoplasts the vegetative nucleus andthe generative cell became tightly surrounded by a new actin network. In the transvacuolar strands there were numerous actin bundles. The “ends” of some of these bundles appeared to be tightly attached to the protoplast membrane indicating that some kind of structures might be present in the protoplast membrane for actin filament attachment.     

Association of the Generative Cell and Vegetative Nucleus in Pollen Tubes of Rhododendron

Mixed fluorescence/bright field microscopy of Rhododendron pollentubes in the first 72 h after germination reveals a lens-shapedgenerative cell which divides to give two associated spermswithin the original cell boundary. The generative cell is closelyassociated with the vegetative nucleus which precedes it in92 per cent of pollen tubes. Three-dimensional reconstruction from serial thin sections ofa pollen tube fixed 24 h after germination shows that the associationbetween the generative cell and vegetative nucleus is extremelycomplex. Elongated tails of the generative cell physically enfoldthe vegetative nucleus and penetrate into enclaves within it.The association has been clarified by use of the periodic acid-phosphotungsticacid-chromic acid technique to enhance electron contrast ofthe plasma membranes surrounding the generative cell. In thisbicellular system, the male germ unit association is apparentlyinitiated after pollen maturity but prior to generative celldivision. Pollen tube, generative cell, male germ unit, plasma membrane, vegetative nucleus, Rhododendron, Ericaceae     

花粉管细胞壁结构及胞质运动

花粉管的极性顶端生长是将雄配子体运输到子房的过程,在高等植物有性生殖过程中起着重要的作用。花粉管的生长过程包括许多方面,其中最为重要的是花粉管细胞壁的合成和胞质运动。本文就细胞壁的结构及组成,生殖细胞和营养核的移位,细胞器以及分泌小泡的运动等方面作了较全面论述。     

Fluorescence Microscopic Observations on Actin Filament Distribution in Corn Pollen and Gladiolus Pollen Protoplasts

Actin filament (AF) distribution in Zea mays pollen and Gladiolus gandavensis pollen protoplasts was localized by FITC conjugated phalloidin fluorescence microprobe. The pollen was incubated in Brewbaker and Kwack (BK) medium, and the pollen protoplasts were isolated enzymatically and cultured in K3 medium containing various supplements by a previously reported method. Samples were fixed for 30 min with 1.5% paraformaldehyde dissolved in 0.1 mol/1 phosphate buffer (pH 7), half strength of BK elements, 1 mol/1 EGTA and sucrose, stained for 30–60 min with 1 μg/ml FITC-phalloidin in the buffer solution, and observed by a fluorescence microscopy. In hydrated corn pollen grains, the AFs constituted an irregular network. Prior to germination a part of the pollen grains showed polarized pattern of Afs. At the opposite pole to the germ pore, there was a center from which AF bundles radiated and converged toward the pore, often making a spindle-shaped configuration. In just isolated gladiolus pollen protoplasts, the AFs appeared as irregular fine network. After 4–7h of culture, the AF distribution coincided in some cases with the unevenly regenerated new wall area as exhibited by FITC-phalloidin and Calcofluor White ST double staining, indicating a possible involvement of AF in wall synthesis. After 17–18 h of culture, a part of the pollen protoplasts went on germination. The AFs became polarized in such protoplasts and converged into the tubes produced, and ran longitudinally along the tubes just like in the tubes germinated from pollen grains. However, in ungerminated pollen protoplasts, the AFs behaved abnormalty, showing various irregular arrangements. When protoplasts bursted, the actin aggregates often located at the protrusion site from which the protoplasts would burst, and were discharged into the medium. In neither corn pollen nor gladiolus pollen protoplasts AFs were observed within the generative or sperm cells.     

The Comportment of the Vegetative Nucleus and Generative Cell in the Pollen and Pollen Tubes of Helleborus foetidus L

It has been reported that in species of Plumbaginaceae, Chenopodiaceae,Cruciferae and Amaryllidaceae a ‘male germ unit’is formed in which the two male gametes remain inter-connected,with one of the pair linked intimately to the vegetative nucleus.In two species the unit has been shown to remain intact in thepollen tube, and some accounts imply that it is polarized inits movement, the vegetative nucleus leading in the tube. Evidence given in this paper indicates that such a unit is unlikelyto be present in Helleborus foetidus L. (Ranunculaceae). Applicationof an optical sectioning technique has shown that at no timeis there a persistent linkage between the generative cell andthe vegetative nucleus in unhydrated, hydrated and germinatingpollen, nor is one present in the early pollen tube. Furthermore,no inter-connections between the two entities were seen in protoplastsfrom living, hydrated and incipiently germinating grains isolatedmechanically in an osmotically balancing medium. Following germination,the vegetative nucleus leaves the grain in advance of the generativecell in most instances, but in the samples examined the generativecell led in about 30 per cent of the tubes. Assembling a polarisedmale germ unit in these circumstances would require (a) theformation of an inter-connection between the vegetative nucleusand the generative cell or one of the gametes derived from itduring passage through the tube, and (b) where the generativecell initially leads in the tube, an exchange in relative positions.It is considered improbable that these conditions could consistentlybe met. Mature, incipiently germinating pollen of H. foetidus releasesa fibrillar component when extruded into suitable media. Websor clusters of fibrils are commonly seen to be associated withboth the vegetative nucleus and the generative cell. The possibilitythat the fibrils are composed of aggregates of microfilamentsis considered. Helleborus foetidus L., pollen germination, generative cell, vegetative nucleus, male germ unit     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

Ultrastructural and Cytochemical Study on Mature Pollen of Pinus tabulaeformis

The pollen of Pinus tabulaeformis Cart. comprised two prothallial cells, a generative cell and a tube cell which degenerated at pollen maturation. The generative cell had its own cell wall, seperating from the intine of pollen, but with its side wall attached to the infine. Cytoplasmic channels were present on the side of the generative cell wall, which faced to the tube cell cytoplasm. The generative cell differed conspicuously from the tube cell. The main differences include: ( 1 ) The chromatin in the generative cell nucleus was condensed, but was dispersed and had numerous nueleare pores in the tube cell nucleus; (2)There was no microbody in the generative cell but many microbodies were present in the tube cell cytoplasm; (3)More inclusions were present in the tube cell than in the generative cell. Both the generative cell and the tube cells contained lipid bodies and amyloplasts in the cytoplasm, but there were more amyloplasts in the former. The tube cell also contained a few proteins which was absent in the generative cell. In addition, there were numerous mitochondria, polyribosomes, and a few endoplasmic reticulums and dictyosomes in the generative and tube cells. DAPI staining demonstrated numerous cytoplasmic DNA in both generative cell and tube cell. The mode of cytoplasmic inheritance, and the composition, structure and the nature of the pollen wall of P. tabulaefonnis are also discussed in this paper.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

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.     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

土麦冬离体萌发花粉管中生殖细胞与营养核的动态变化

主要报道了土麦冬人工培养萌发花粉管中生殖细胞与营养核的动态变化。多数花粉管中,生殖细胞与营养核贴合后,开始进行有丝分裂,贴合时,营养核略呈弥散状态。在分裂早中期,生殖细胞与营养核分开,从贴合到分开大约经历３－５ｈ,精子形成后,不与营养核连接。ＤＡＰＩ对生殖细胞的有丝分裂有抑制作用。少数花粉管中,生殖细胞核进行无丝分裂,有缢裂和劈裂两种方式。生殖细胞核发生缢裂的花粉管中,未观察到生殖细胞与营养核的贴     

Membrane potential changes during pollen germination and tube growth

Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC₄(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (−108 mV) with respect to the pollen grains (−23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H⁺-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H⁺-ATPase and anion channels in the regulation of its value.     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.