花粉管细胞结构与生长机制研究进展

花粉管的极性顶端生长是一个复杂的动力学过程, 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骨架动态和胞质运动。本文较全面地综述了花粉管的结构、细胞骨架、胞质运动、囊泡转运及循环、线粒体运动以及内质网和高尔基体之间囊泡运动等。     

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。     

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。     

多头绒泡菌中动蛋白的免疫化学鉴定

动蛋白(kinesin)是一种微管系统的运动蛋白(motor protein),它能通过水解ATP将化学能转化为机械能,推动微管产生运动.微管系统作为一种主要的细胞骨架存在于所有真核细胞中,它们对于维持细胞形态,细胞的分裂,染色体的运动及细胞内的物质运输起着重要作用.细胞质力蛋白(dynein)和动蛋白是公认的推动这类运动的运动蛋白.自从1985年Vole首次在鱿鱼大轴突(squidgiant axon)中发现动蛋白以来,人们先后在许多种动物细胞中发现有动蛋白存在,甚至在低等真核生物棘状变形虫,盘基网柄茵和高等植物烟草花粉管中发现有动蛋白的存在.研究结果表明,动蛋白参与了真核细胞中的许多重要生命活动,如细胞中的细胞器及囊泡的运动,染色体排裂和分离等运动.动蛋白很可能是普通存在于所有真核细胞中的一种运动蛋白.多头绒泡菌(Physarum poly-cephalum)属于粘菌纲(Myxomycetes)的一种低等真核生物,它表现出许多显著的细胞运动特征如原生质团迁移,细胞质的穿梭运动(shuttle streaming)等,是研究非肌细胞运动和收缩蛋白的经典材料.在多头绒泡菌胞质中也具有微管系统,它们构成其纺锤丝等,参与染色体的运动及其它胞质运动,但至今国内外尚无人证明其中有与微管作用的运动蛋白——动蛋白的存在,作者利用抗牛脑动蛋白的单克隆抗体,     

细胞骨架蛋白调节囊泡转运及其与神经疾病的关系

细胞内囊泡转运依赖于细胞骨架系统,细胞骨架为囊泡转运提供了轨道,而细胞骨架表面的马达蛋白则为其提供了动力。近年来,随着活细胞成像技术以及相关的生化、药理实验方法的不断进步,人们对囊泡转运的分子机制有了更加深入的认识。越来越多的实验结果表明,细胞骨架蛋白对囊泡转运有着重要的调节作用。囊泡转运的紊乱与多种神经疾病相关。囊泡转运分子调控机制的研究,将为多种神经疾病的治疗提供新的思路。     

NtGNL1基因通过调控囊泡运输影响烟草根毛的极性生长

极性生长是植物生长发育中的常见现象,但囊泡运输与极性生长的关系还未完全明确。花粉管和根毛是植物细胞极性生长的典型模式。早期研究显示NtGNL1(Nicotiana tabacum GNOM-LIKE 1)通过调节囊泡的后高尔基体转运来影响烟草的花粉管生长。本文以NtGNL1 RNAi转基因植株为材料,研究NtGNL1基因在根毛生长中的作用。结果表明,NtGNL1 RNAi转基因植株的根毛生长明显滞后于野生型,且其根毛出现膨大、弯折、扭曲等形态,与NtGNL1 RNAi转基因植株的花粉管异常形态类似。q RT-PCR检测RNAi转基因株系根毛中PIN1、PIN2、GL2、ROP6、RHD6基因的m RNA表达量,显示PIN2和GL2的表达量显著下调,PIN1、ROP6和RHD6的表达量变化不明显。FM4-64染色表明烟草根表皮细胞和根毛的囊泡分布都受到影响,即NtGNL1基因也影响根毛中的囊泡运输。BFA处理加剧了囊泡的聚集程度,提示根毛尖端还存在其它对BFA敏感并调控囊泡运输的基因。以上证据显示,NtGNL1基因通过囊泡运输途径影响烟草根毛的极性生长,NtGNL1基因的表达下调也影响了PIN2和GL2的表达,从而间接影响根毛的极性生长。     

微丝骨架的构成及其对花粉管极性生长的调控作用

微丝骨架是细胞骨架的重要组成部分,它由肌动蛋白和肌动蛋白结合蛋白组成,广泛存在于真核细胞中。近年来,大量研究表明植物花粉及花粉管中存在丰富的微丝骨架。目前,在微丝骨架作为信号转导途径的靶标参与对花粉管极性生长的调控、微丝骨架在花粉和花粉管中的分布及其在花粉管生长过程中与其他信号分子之间的相互作用等方面取得了一系列突破性进展。     

花粉管生长调控的研究进展

本文从花粉管的生长特性、细胞质组成、细胞骨架、细胞壁的结构与合成、Ｃａ２＋通道和向性生长机制六个方面,综述了近些年来对花粉管生长调控研究的进展。     

生物膜的动态结构和功能——从二维到三维

生物膜上蛋白质的运动不都是随机性的,经常受到细胞骨架等因素的限制。二维膜上的事件不断通过内吞、分泌等过程和整体细胞的功能联系起来。病毒感染细胞、细胞内囊泡和物质的定向运输等都和细胞内分子的识别、膜的融合和膜的分裂等过程紧密相关。     

膜联蛋白: 植物生长过程中的多功能复合物

该文介绍了植物膜联蛋白(annexins)以及在生长过程中不同生理活动所扮演的角色,如钙离子通道的形成、膜融合、囊泡运输、信号转导和细胞骨架蛋白间的相互作用,以及可以结合F-肌动蛋白,具有过氧化物酶、离子通道,使ATP和GTP水解的功能。     

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。     

Lead Stress Disrupts the Cytoskeleton Organization and Cell Wall Construction During <Emphasis Type="Italic">Picea wilsonii</Emphasis> Pollen Germination and Tube Growth

Lead is a widespread pollutant and has been reported to inhibit pollen tube development, but the mechanism of toxicity involved remains unclear. Here, we report that lead stress significantly prevented Picea wilsonii pollen germination and tube growth and also dramatically altered the tube morphology in a concentration-dependent manner. Fluorescence labeling with JIM 5 (anti-acidic pectin antibody) and Calcofluor white revealed the lead-induced decline of acidic pectin and cellulose, especially in the subapical region. Decolorized aniline blue staining showed the marked accumulation of callose in the apical and subapical regions of lead-treated tubes. Fluorescence labeling with Alexa Fluor 568 phalloidin and anti-tubulin antibody revealed that the distribution of the cytoskeleton in P. wilsonii pollen grains and tubes were developmentally regulated and that lead disturbed the cytoskeleton organization, especially in the shank of the pollen tubes. Taken together, our experiments revealed a link between the dynamics of cytoskeleton organization and the process of P. wilsonii pollen tube development and also indicated that lead disturbed the cytoskeleton assembly and, consequently, cell wall construction. These findings provide new insights into the mechanism of lead toxicity in the tip growth of pollen tubes.     

Actin bundler PLIM2s are involved in the regulation of pollen development and tube growth in Arabidopsis

Microspores develop inside the anther, where they are surrounded by nourishing tapetal cells. However, many cellular processes occurring during microspore development in the locule are poorly characterized. The actin cytoskeleton is known to play a crucial role in various aspects of the plant developmental process. During pollen tube tip growth, actin cytoskeleton serves as an efficient molecular transportation track, although how it functions in pollen development is unknown. The plant actin bundler PLIM2s have been shown to regulate actin bundling in different cells. Here, we investigate the biological function of three Arabidopsis pollen-specific LIM proteins, PLIM2a, PLIM2b, and PLIM2c (collectively, PLIM2s), in pollen development and tube growth. Variable degrees of suppressed expression of the PLIM2s by RNA interference resulted in aberrant phenotypes. Complete suppression of the PLIM2s totally disrupted pollen development, producing abortive pollen grains and rendering the transgenic plants sterile. Partial suppression of the PLIM2s arrested pollen tube growth to a lesser extent, resulting in short and swollen pollen tubes. Finally, the PLIM2c promoter initiated expression in pollen during stamen filament elongation, and the PLIM2c protein was located on particle structures in the developing pollen grains in Arabidopsis. These suggest that the actin bundler, PLIM2s, are an important factor for Arabidopsis pollen development and tube growth.     

Rop1Ps promote actin cytoskeleton dynamics and control the tip growth of lily pollen tube

Rop, the small GTPase of the Rho family in plants, is believed to exert molecular control over dynamic changes in the actin cytoskeleton that affect pollen tube elongation characteristics. In the present study, microinjection of Rop1Ps was used to investigate its effects on tip growth and evidence of interaction with the actin cytoskeleton in lily pollen tubes. Microinjected wild type WT-Rop1Ps accelerated pollen tube elongation and induced actin bundles to form in the very tip region. In contrast, microinjected dominant negative DN-rop1Ps had no apparent effect on pollen tube growth or microfilament organization, whereas microinjection of constitutively active CA-rop1Ps induced depolarized growth and abnormal pollen tubes in which long actin bundles in the shank of the tube were distorted. Injection of phalloidin, a potent F-actin stabilizer that inhibits dynamic changes in the actin cytoskeleton, prevented abnormal growth of the tubes and suppressed formation of distorted actin bundles. These results indicate that Rop1Ps exert control over important aspects of tip morphology involving dynamics of the actin cytoskeleton that affect pollen tube elongation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

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.     

Investigating mechanisms involved in the self-incompatibility response in Papaver rhoeas

Sexual reproduction in flowering plants is controlled by recognition mechanisms involving the male gametophyte (the pollen) and the female sporophyte (the pistil). Self-incompatibility (SI) involves the recognition and rejection of self- or incompatible pollen by the pistil. In Papaver rhoeas, SI uses a Ca(2+)-based signalling cascade triggered by the S-protein, which is encoded by the stigmatic component of the S-locus. This results in the rapid inhibition of incompatible pollen tube growth. We have identified several targets of the SI signalling cascade, including protein kinases, the actin cytoskeleton and nuclear DNA. Here, we summarize progress made on currently funded projects in our laboratory investigating some of the components targeted by SI, comprising (i) the characterization of a pollen phosphoprotein (p26) that is rapidly phosphorylated upon an incompatible SI response; (ii) the identification and characterization of a pollen mitogen-activated protein kinase (p56), which exhibits enhanced activation during SI; (iii) characterizing components involved in the reorganization and depolymerization of the actin cytoskeleton during the SI response; and (iv) investigating whether the SI response involves a programmed cell death signalling cascade.     

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.     

Signals and targets of the self-incompatibility response in pollen of Papaver rhoeas

Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in inhibition of incompatible pollen. A picture of some of the signalling events and mechanisms involved in this specific inhibition of pollen tube growth is beginning to be built up. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. Rapid increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) can now be attributed (at least in part) to Ca(2+) influx. The rapid loss of the pollen apical Ca(2+) gradient within approximately 1-2 min is accompanied by the inhibition of pollen tube tip growth. Concomitant with this time-frame, hyper-phosphorylation of p26, a soluble pollen phosphoprotein is detected. Characterization of p26 reveals that it is a soluble inorganic pyrophosphatase, which suggests a possible direct functional role in pollen tube growth. Slightly later, a putative MAP kinase (p52) is thought to be activated. Finally, preliminary evidence that programmed cell death (PCD) may be triggered in this response is described. A key target for these signals, the actin cytoskeleton, has also been identified. In this article the current understanding of some of the components of this signalling cascade and how they are beginning to throw some light on possible mechanisms involved in this SI-induced inhibition of pollen tube growth, is discussed.     

高等植物自交不亲和反应中花粉管信号传导研究进展(综述)

高等植物自交不亲和反应是由基因控制、避免发生自花授粉的一种机制。本文介绍以虞美人为主的高等植物在自交不亲和反应中肌动蛋白骨架的动态变化及Ca2 的时空变化,着重阐述花粉管生长被抑制的最初信号传导。