应用水稻花粉离体萌发体系观察花粉管内微丝分布

采用非固定、DMSO渗透和异硫氰酸标记的鬼笔环肽（FITC—Ph）染色方法,观察水稻花粉离体萌发过程中花粉管内肌动蛋白微丝的形态和分布。结果表明：（1）水稻花粉水合2min后即可萌发,花粉管生长速度在600～1500μm／h之间。（2）水合而未萌发的花粉粒中,大量较短的梭形微丝束构成微丝网络结构,萌发过程中花粉粒内的梭形微丝束松解,部分微丝转移至萌发的花粉管内沿花粉管纵轴呈束状结构;随着花粉管的伸长,微丝束主要分布在花粉管中前端,但在花粉管顶端区域始终未见明显的微丝束。（3）水合后不能正常萌发的花粉粒内肌动蛋白微丝呈弥散不规则分布,在相同萌发时间生长迟缓的花粉管中,微丝束较少,且主要位于花粉管近萌发孔的部位。表明微丝骨架的形态和分布影响水稻花粉管的萌发和生长。     

肌动蛋白在丝瓜花粉管顶端生长中的作用

用非固定荧光标记的鬼笔环肽作为肌动蛋白探针观察并证明了丝瓜未萌发的花粉粒和不同生长时期花粉管中肌动蛋白纤丝的分布及其形态变化。又用细胞松弛素B（CB）、氯两嗪（CPZ）及N-乙酰马来酰胺（NEM）证明了丝瓜花粉管伸长与肌动蛋白既有密切的关系,也受Ca2 的调节。     

肌动蛋白在丝瓜花粉管顶端生长中的作用

用非固定荧光标记的鬼笔环肽作为肌动蛋白探针观察并证明了丝瓜未萌发的花粉粒和不同生长时期花粉管中肌动蛋白纤丝的分布及其形态变化。又用细胞松弛素Ｂ（ＣＢ）、氯丙嗪（ＣＰＺ）及Ｎ－乙酰马来酰胺（ＮＥＭ）证明了丝瓜花粉管伸长与肌动蛋白暨有密切的关系，也受Ｃａ＾２＋的调节。     

在烟草和蓝猪耳花粉萌发及花粉管生长中阿拉伯半乳糖蛋白的作用

采用花粉离体培养技术研究了阿拉伯半乳糖蛋白(arab inogalactan prote ins,AGPs)在烟草和蓝猪耳花粉萌发及花粉管生长中的作用。结果表明:βG lcY(-βG lucosyl Yariv reagent,一种能与AGPs特异结合的试剂)处理导致蓝猪耳花粉萌发率和花粉管生长速度下降;βG lcY对烟草花粉管的生长也有一定的抑制作用。另外,采用免疫荧光技术,发现在烟草和蓝猪耳未萌发的花粉粒中,AGPs主要分布在萌发孔上;在烟草离体生长的花粉管中AGPs呈均匀分布,但是在蓝猪耳离体生长的花粉管中未观察到AGPs的分布。     

凝溶胶蛋白对植物微丝的调节作用及在花粉中的定位

以植物花粉为实验材料, 研究了来源于动物的凝溶胶蛋白对植物肌动蛋白丝的作用及凝溶胶蛋白在植物细胞中的定位. 利用离心沉淀丝状肌动蛋白及电子显微镜负染的实验结果显示, 花粉纯化肌动蛋白丝及粗提液中肌动蛋白丝可被凝溶胶蛋白剪切, 而且这种剪切作用依赖于Ca²⁺. 另外, 利用免疫沉淀测定凝溶胶蛋白与花粉肌动蛋白单体结合的实验表明, 当溶液中有Ca²⁺. 存在时, 花粉肌动蛋白与凝溶胶蛋白的摩尔比值约为2.0 ± 0.21; 用EGTA去除Ca²⁺. 后, 该比值有所降低, 减至1.2 ± 0.23; 而加入PIP2后, 这种结合比值又降至0.8 ± 0.10, 表明植物肌动蛋白与凝溶胶蛋白的结合有类似于动物肌动蛋白的特性, 受到Ca²⁺. 和PIP2的调节. 花粉中凝溶胶蛋白的免疫鉴定及免疫荧光定位实验结果表明, 花粉中存在凝溶胶蛋白, 在未萌发的花粉粒中凝溶胶蛋白主要集中在萌发沟处, 而在萌发2 h的花粉管中, 花粉管胞质内均可看到荧光分布, 但花粉管顶端荧光较强, 推测凝溶胶蛋白可能参与花粉的萌发和花粉管的生长.     

月见草花粉萌发后花粉管和花粉粒内细胞器的运动

采用电视显微镜观察了月见草花粉萌发后花粉管和花粉粒内的细胞器运动,对细胞器运动过程进行了录相,并用计算机对运动图象作了分析。结果表明,月见草花粉内细胞器运动有连续运动和不连续运动两种形式;细胞器运动不依赖于细胞质流动。时快时慢,而且是单向的,没有观察到细胞器的双向运动,细胞松驰素Ｂ能抑制细胞器运动,在萌发的花粉粒内还十分清晰地观察到纤丝束摆动,细胞器沿纤丝运动。     

紫萼花粉粒和花粉管中微丝的超微结构

用常规化学固定和化学固定前用鬼笔环肽处理两种电镜样品制作技术,分别研究了紫萼[Hosta venteicosa (=H.coerulea]成熟花粉粒和幼花粉管中的微丝的超微结构。结果表明,在常规电镜固定中花粉粒中的微丝能保存,但在花粉管中的则遭受破坏。用鬼笔环肽处理后化学固定的方法,微丝在花粉管中能良好地保存。在花粉粒中平行的微丝形成束,表现为具分布的特点,即限于分布在它们功能的区域,并且微丝束经常紧密地与营养核贴近。在幼花粉管中微丝束表现为在线粒体、质体、内质网、小泡和小液泡的表面通过,并常常与脂体紧密联结。这些现象表明在花粉萌发和花粉管生长时,微丝与营养核及与其它细胞器的运动之间存在某些联系的迹象。     

花粉粒和花粉管中的微丝骨架

自从1972年Franke等第一次报道大君子兰和麝香百合花粉管中存在直经6nmn的肌动蛋白微丝以来,近十几年来这方面的研究工作已迅速展开,积累了丰富的研究资科。借助于荧光探针、免疫荧光标记、透射电镜等技术手段,已先后对三十几种植物的花粉粒和(或)花粉管的微丝做过观察,揭示了花粉萌发和花粉管生长过程中肌动蛋白微丝的三维结构及其在时间和空间上变化的规律,并对微丝在花粉萌发和花粉管生长的生理活动中的重要作用获得了比较一致的认识。     

朱顶红花粉管中营养核的纤丝状内含体

用透射是镜研究了朱顶红生殖细胞发育过程中花粉管的营养核中的纤丝状内含体的结构形态。此内含体在花粉管生长早期即可观察到,并在生殖细胞分裂过程中频繁发现。它们具各种方向,未见与其它细胞结构发生联系。通过比较此内含体与花粉管中肌动蛋白纤丝的形态及两者对细胞松弛素Ｄ（ＣＤ）处理的反应,我们倾向认为营养核的纤丝状内含体可能由肌动蛋白组成。讨论了此结构在建立营养核运动机制上的作用。     

花粉萌发转录基因组研究

为了解花粉萌发的详细细节,中国科学家利用拟南芥基因组芯片,首次发现从脱水成熟花粉粒,到含水花粉粒,再到拟南芥的花粉管的转录组变化。在花粉萌发和花粉管生长过程中,与细胞存活、转录、信号传导和细胞转运有关的表达基因发生了很大变化,尤其是那些正调控的基因。此外,类钙调蛋白、阳离子交换因子、以及热激蛋白家族在花粉萌发和花粉管生长过程中变化较大,     

Visualization of Actin Filament Patterns in Pollen Tubes of Hosta caerulea Tratt. with a Non-Fixation and TRITC-Phalloidin Method

Actin filament patterns during pollen germination in Hosta caerulea Tratt. were visualized with a simple method in which there was no pre-fixation, with dimethylsulphoxide (DMSO) as a permeabilising agent and staining with TRITC-Phalloidin. The cytoplasm of the vegetative cell of the ungerminated pollen grain contained numerous crystalline fusiform bodies to constitute a storage form of actin. These bodies were transferred to the emerging pollen tube after the germination of the pollen grain. Following the growth of pollen tube, the fusiform bodies were gradually dissociated, branched, slenderized and formed a cross-linked actin network. During the further growth of the pollen tube, the preponderance of longitudinally-oriented thin actin filaments with some anastomoses to form a more complex network present always in the long pollen tube. This was the typical pattern of actin filaments in most cases. In some conditions, actin filaments were assembled to form thick actin cables near the proximate part of the pollen tube tip. The branching and connecting of the cables were probably also seen in some parts. Actin filaments were always entering to the apical region of a tube tip. The significance of the non-fixation and fluorescence-phalloidin (FI-Ph) method and the problems in the future studies are discussed     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

Pyrus pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen and tubes in vitro

Summary. Pears (Pyrus pyrifolia L.) have an S-RNase-based gametophytic self-incompatibility system, and S-RNases have also been implicated in self-pollen or genetically identical pollen rejection. Tip growth of the pollen tube is dependent on a functioning actin cytoskeleton. In this study, configurations of the actin cytoskeleton in P. pyrifolia pollen and effects of stylar S-RNases on its dynamics were investigated by fluorescence and confocal microscopy. Results show that actin filaments in normal pollen grains exist in fusiform or circular structures. When the pollen germinates, actin filaments assembled around one of the germination pores, and then actin bundles oriented axially throughout the shank of the growing tube. There was a lack of actin filaments 5–15 μm from the tube tip. When self-stylar S-RNase was added to the basal medium, pollen germination and tube growth were inhibited. The configuration of the actin cytoskeleton changed throughout the culturing time: during the first 20 min, the actin configurations in the self-pollen and tube were similar to the control; after 20 min of treatment, the actin filaments in the pollen tube gradually moved into a network running from the shank to the tip; finally, there was punctate actin present throughout the whole tube. Although the actin filaments of the self-pollen grain also disintegrated into punctate foci, the change was slower than in the tube. Furthermore, the alterations to the actin cytoskeleton occurred prior to the arrest of pollen tube growth. These results suggest that P. pyrifolia stylar S-RNase induces alterations in the actin cytoskeleton in self-pollen grains and tubes. Correspondence: Shao-ling Zhang, College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China.     

Spatial and temporal organization of actin during hydration,activation, and germination of pollen inPyrus communis L.: a population study

Summary Dynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.     

Germination of Monocolpate Angiosperm Pollen: Evolution of the Actin Cytoskeleton and Wall during Hydration, Activation and Tube Emergence

The monocolpate pollen grain of Narcissus pseudonarcissus L.has two preferred sites for tube emergence, one at each endof the colpus. While the cellulosic microfibnls of the innerlayer of the intine are disposed circumferentially in the centreof the grain, the microfibrils in these terminal sites are shorterand randomly oriented Soon after the beginning of hydration,inclusions of the vegetative cell begin movement, firstly ina rotatory manner, and then in a pattern focused on one or bothgermination sites, where the intine bulges as hydration progresses.These changes are associated with the evolution of the actincytoskeleton. Actin is present in the unactivated grain in theform of fusiform bodies. During hydration these dissociate toform finer fibrils, initially randomly disposed. Then, correlatedwith the change of the pattern of movement in the vegetativecell, the actin fibril system becomes polarized towards thegermination sites, where shorter fibrils accumulate. Callose,absent from the ungerminated grain, is deposited within thecellulosic wall in these locations, forming a shallow dome whicheventually develops into an annulus subtending the inner calloselining of the emerging tube. The transition to cylindrical growthis associated firstly with the development of zonation in thecytoplasm of the vegetative cell, with the tip occupied by apopulation of wall precursor bodies (P-particles) and a denseaggregate of short actin fibrils; and then with the establishmentof the ‘inverse fountain’ pattern of movement characteristicof the apical part of the extending tube. Narcissus pseudonarcissus L, pollen activation, pollen germination, actin cytoskeleton, tip-growth system, pollen-tube wall development     

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

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

微丝骨架动态参与花粉萌发的研究

利用改进的Alex-phalloidin活细胞染色方法和激光共聚焦显微镜技术,观察川百合(Lilium davidii Duch)花粉原生质体极性形成及萌发过程中微丝骨架的列阵变化.结果表明,花粉原生质体从贮存状态,经过水合、极性形成至萌发花粉管,其微丝结构从短小的梭形体,经过形成均匀的网状结构、向细胞边缘汇集的平行排列的束状结构,逐渐变成多层连续环绕细胞的微丝束结构.用酪氨酸磷酸酶抑制剂苯胂化氧(PAO)处理花粉原生质体,在微丝的汇合处,肌动蛋白聚集成小的团块,花粉的萌发受到抑制;而利用酪氨酸磷酸激酶抑制剂genistein处理细胞,微丝结构的列阵变化与对照相似.结果说明,在川百合花粉萌发过程中,有某种酪氨酸磷酸酶参与了反应.     

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.