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。     

用荧光染色技术标记生活花粉管的研究

在金鱼草和(或)烟草上试验了三种荧光染料对花粉管进行荧光活体染色与标记的效果。花粉管在异硫氰酸荧光素(FITC,12.5μg/ml)中染色5—6小时,原生质呈绿黄色荧光;换入无染料的培养基后可继续生长并保持荧光标记,但后期生长受抑。罗丹明B(RB,10μg/nl)染色的效果与上相近,花粉管呈红色荧光;换入无染料培养基后生长正常,唯后期荧光减弱。荧光素二醋酸酯(FDA,10μg/ml)染花粉粒40分钟,换入无染料培养基后正常萌发与生长,花粉管原生质呈明亮的绿色荧光。FDA方法具有染色时间短,荧光明亮,兼有活染与生活力鉴定双重功效等优点。     

植物花粉管中类整联蛋白的免疫荧光定位研究

利用免疫荧光技术,分别用整联蛋白β_3、α_y亚基胞质域抗血清及整联蛋白VnR(Vitronectin的受体)的抗血清在百合花粉管中检测出荧光标记,且在花粉管尖端逐渐增强,而用FnR(纤粘连蛋白的受体)、LnR(层粘连蛋白的受体)的抗血清及非免疫血清对照实验在百合花粉管中则没有或仅有很弱的荧光。在萌发的碧桃花粉管中,用β_1、β_3亚基胞质域抗血清也能检测出荧光标记,初步结果显示花粉管在生长过程中,质膜上有可能存在类整联蛋白,并且在百合花粉管中这种类整联蛋白可能是由α_v和β_3两种亚基组成的Vn样蛋白的受体。     

花粉管钙信号特性及其调控研究进展

花粉管在花柱中生长受多个信号分子的协同调控,钙离子在其中发挥着重要作用.钙是一种重要的第二信使,它将外界的多种生物或非生物信息转化为对细胞内基因表达以及细胞生理反应的调控.钙信号表达方式是胞内自由钙浓度的特异性变化.该文对国内外近年来有关花粉管生长中钙信号特性及其调控的研究进展,如花粉管尖端自由钙离子浓度梯度与胞内钙振荡、花粉管质膜钙转运体的鉴定及其调控特性、花粉管钙信号与微丝和ROP蛋白的关系以及花粉管钙信号与植物自交不亲和性反应的关系等进行综述,为深入开展相关研究提供参考.     

百合远缘杂交花粉萌发及花粉管生长过程观察

利用荧光显微镜对百合远缘杂交组合Bernini×Pollyanna花粉萌发及花粉管生长过程进行观察研究,结果显示,授粉后3~30 h内花粉萌发形成花粉管,且花粉管生长速度由快到慢,48~51 h内花粉管停止生长,花粉管最后深入到花柱的1/3处,并观察到一些异常的花粉管形态;花粉管生长过程中还伴随着一系列的胼胝质反应,出现的部位依次是乳突细胞、花粉管、花柱通道细胞、胚珠中的胚囊.结果表明该杂交组合不亲和.研究认为Berni-ni柱头乳突细胞是阻碍Pollyanna花粉管生长的第一道屏障,花柱通道细胞是抑制花粉管在花柱内生长的第二道屏障.     

毛竹花粉的形态及双受精过程研究

由于实验的材料、条件和技术的限制,关于竹类花粉形态、花粉管生长过程的研究十分少见。毛竹( Phyllostachys edulis)属于禾本科( Poaceae/Gramineae)竹亚科( Bambusoideae)植物,在我国广泛分布,具有重要的经济、生态和文化价值。由于其独特的生殖生物学特性,即开花周期长、结籽率低,所以对其开花材料的获取十分困难。该研究在其独特的开花特性基础上,结合野外实地观察,利用显微镜、环境扫描电镜、荧光显微镜等,对毛竹的花粉形态、结构以及花粉管的萌发与生长动态进行了首次报道。结果表明：(1)毛竹花粉近球形,平均直径为61.00μm,属于大型孢子。(2)毛竹成熟花粉落到雌蕊柱头上后,经过花粉与柱头的成功识别,开始萌发,并形成花粉管;随着花粉管的逐渐伸长生长,依次经过柱头和花柱,到达子房;约120 min后到达珠孔处,进入胚珠和胚囊,完成双受精作用。通过对毛竹花粉形态结构和花粉管生长至受精过程的动态观察,为毛竹花粉可授性、花粉生理、生化研究提供了实验技术准备。该研究结果完善和丰富了毛竹乃至整个禾本科植物在生殖生物学方面的研究内容。     

内钙拮抗剂TMB-8对白皮松花粉管细胞壁构建的影响

应用荧光显微技术、激光共聚焦扫描显微技术、单克隆抗体免疫荧光标记技术以及傅里叶变换显微红外光谱分析(FTIR)等手段,研究了内钙拮抗剂TMB-8对白皮松花粉管胞内Ca2+分布、花粉管生长以及细胞肇构建等的影响.结果表明,白皮松花粉管经TMB-8处理后,胞内的Ca2+浓度下降,花粉管内典型的Ca2+浓度梯度消失,花粉萌发...     

FITC示踪在优化小麦花粉管通道转化方法中的应用

利用FITC(fluorescein is othiocyanate,异硫氰酸荧光素)标记的外源DNA对小麦进行了花粉管通道法转化,在荧光显微镜下观察了外源DNA进入小麦胚囊的情况,并初步研究了转化时间及转化溶液组成对DNA进入胚囊效率的影响。结果表明,外源DNA沿着花粉管生长过程中形成的花粉管通道进入胚囊;授粉45 min和60min进行转化外源DNA进入胚囊的几率较大,因此在此段时间开始转化较为合适;相对于TE缓冲液,将0.05%Silwet L-77和5%蔗糖作为转化溶液可以缩短DNA进入胚囊的时间,但不能提高外源DNA进入胚囊的几率。研究表明,使用FITC标记观察外源DNA进入胚囊效率的方法,可简便有效地应用于花粉管通道法转化条件的初步优化。     

持续激活型CIPK9在花粉管生长过程中的生物学功能及亚细胞定位分析

植物的花粉管生长是一个多因素参与的生理学过程,需要多种信号传导系统来引导植物细胞完成。钙离子作为第二信使,可以通过钙传感器CBLs激活下游的蛋白激酶CIPKs参与调控细胞的极性发育过程。该研究中 CIPK9 被确定为候选基因,其C端与绿色荧光蛋白(GFP)相融合,通过基因枪技术在烟草花粉中进行瞬时表达,观察对应的亚细胞定位及花粉管中诱导的表型。结果表明:(1)GFP标记的CIPK9定位于花粉管中高速运动的颗粒状细胞器,并可随胞质环流进行规律的运动,为进一步探究CIPK9的生物学功能,还构建了持续激活型CIPK9(CACIPK9)。(2)与全长CIPK9相比较,CACIPK9缺少C末端的调控区域,并在激酶区域的激活环中进行了点突变,从而表现出不受调控的持续高活性。(3)缺少C端调控区的CACIPK9表现出非特异性的亚细胞定位,即与GFP对照相同的胞内弥散定位,说明CIPK9的C末端调控区对于其在花粉管中的正确定位发挥重要的调控作用。另外,CACIPK9过表达可以引起花粉管的去极化生长表型。这表明CIPK9作为钙信号下游家族的一员参与了花粉管极性生长的相关过程,并对花粉管的生长具有一定的调控作用。     

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.     

Reproductive lability in pre-dispersal biology ofRhamnus alaternus L. (Rhamnaceae)

Summary Rhamnus alaternus L. is a dioecious, fleshy-fruited shrub, typical of Mediterranean vegetation, which has been recorded from the region since the tropical Tertiary. Reproductive biology of this species has been studied in Southern Italy over a period of 4 years with both field and laboratory experiments. In all the studied populations male plants predominate over the females, have a higher flower density and flower regularly every year while females are usually alternate-bearing. A strong negative association between fruit formation and vegetative growth has been found indicating that the presence of fruits on a branch reduces the growth of new shoots. This phenomenon, together with alternate-bearing, is evidence of a strong competition between vegetative growth and reproductive activity and indicates resource limitation. Flower morphology and quantity of pollen produced are typical of insect pollinated species, and flies of the Empididae and Muscidae were trapped bearing pollen at female plants but pollen is also dispersed by wind. Field experiments on pollination success showed that the exclusion of insects as pollen vectors produces a significant decrease in fruit development. Excess pollen provided by hand-pollination increases fruit set over open-pollination and reproductive success in the field is therefore pollen limited. The variability of sex expression, male fitness, fruit production, regrowth and pollination presented here suggests that these predispersal characteristics ofR. alaternus have been subject to evolutionary change since the tropical Tertiary climate was replaced by a Mediterranean one. This points to the possibility that reproductive, as well as morphological and physiological characteristics, may be equally selected for in extreme environments such as the Mediterranean.     

Pollen vegetative cell-specific expression of Bra r 1: useful tool for observation of the vegetative nucleus and identification of transgenic pollen by nuclear-targeted GFP

Bra r 1 encodes a novel Ca²⁺-binding protein specifically expressed in pollen and is localized in cytoplasm of pollen and pollen tubes. In this study, we demonstrated the expression of green fluorescent protein (GFP) with a nuclear localization signal under the control of Bra r 1 promoter in tobacco pollen. A fluorescent signal was detected in the vegetative nucleus (VN) but not in generative and sperm cell nuclei, indicating pollen vegetative cell-specific expression of Bra r 1. The fluorescent signal in elongating pollen tubes was stronger than that in mature pollen, indicating that the expression of Bra r 1 was more activated during pollen tube growth. This result suggests that Bra r 1 protein might be necessary for pollen tube growth. The pattern of green fluorescence in the VN revealed that VN chromatin is dispersed during the mid-bicellular pollen stage and condensed at the mature stage. This suggests that the level of chromatin condensation might be linked with gene expression in pollen vegetative cells. We also found that the expression of GFP and its targeting of the VN have no detrimental effect on pollen maturation and pollen tube growth. Expression of GFP in pollen thus makes rapid non-destructive monitoring of transgenic pollen and pollen tubes possible. The GFP which moved into the VN was found to be a convenient tool for observation of the VN and could be useful as a selectable marker of transgenic pollen for the analysis of pollen-specific genes. Received: 6 December 2000 / Revision accepted: 20 March 2001     

The Role of Calcium in the Fertilization Process in Flowering Plants

The fertilization process in flowering plants in a broad sense includes a progamic phase preceding the phase of double fertilization. To our present knowledge, calcium as a second messenger in the signal transduction plays important roles in all the links of this process. The present review attempts to highlight the recent advances in this research field, including: calcium in relation to in vitro pollen tube growth (distribution of calcium in pollen tube tip; regulation of pollen tube growth by calcium; calcium oscillation in pollen tube); distribution of calcium in pistil and its relation to in vivo pollen tube growth (calcium in relation to pollen-pistil recognition; pollen tube growth in pistil; pollen tube entry into embryo sac and the discharge and transportation of sperms); and calcium in relation to sperm-egg fusion and egg cell activation. In conclusion the author summarizes into several main view points, and gives recommendation for further researches on this topic.     

钙在被子植物受精过程中的作用

近年来,花粉管中的钙信号和生理功能的研究取得了明显的进展,同时在雌蕊系统中有关钙分布的研究也初步显示了其时、空特征与被子植物的受精作用密切相关。该文总结了花粉萌发和花粉管生长过程中外源钙和内源钙的作用机制,结合雌蕊组织中钙分布的特征,进一步探讨了钙在被子植物受精过程中的功能。     

Pollen performance before and during the autotrophic-heterotrophic transition of pollen tube growth

For species with bicellular pollen, the attrition of pollen tubes is often greatest where the style narrows at the transition between stigmatic tissue and the transmitting tissue of the style. In this region, the tubes switch from predominantly autotrophic to predominantly heterotrophic growth, the generative cell divides, the first callose plugs are produced, and, in species with RNase-type self-incompatibility (SI), incompatible tubes are arrested. We review the literature and present new findings concerning the genetic, environmental and stylar influences on the performance of pollen before and during the autotrophic-heterotrophic transition of pollen tube growth. We found that the ability of the paternal sporophyte to provision its pollen during development significantly influences pollen performance during the autotrophic growth phase. Consequently, under conditions of pollen competition, pollen selection during the autotrophic phase is acting on the phenotype of the paternal sporophyte. In a field experiment, using Cucurbita pepo, we found broad-sense heritable variation for herbivore-pathogen resistance, and that the most resistant families produced larger and better performing pollen when the paternal sporophytes were not protected by insecticides, indicating that selection during the autotrophic phase can act on traits that are not expressed by the microgametophyte. In a study of a weedy SI species, Solanum carolinense, we found that the ability of the styles to arrest self-pollen tubes at the autotrophic-heterotrophic transition changes with floral age and the presence of developing fruits. These findings have important implications for selection at the level of the microgametophyte and the evolution of mating systems of plants.     

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.     

Ion dynamics and its possible role during in vitro pollen germination and tube growth

Summary One of the most interesting aspects of plant fertilization is the growth and orientation of the pollen tube from the stigma to the ovary. Considerable research has been carried out in this field but little is yet known about the mechanisms involved in the growth process. Recent research has been focused on the regulation of molecular events in order to discover the specific genes involved in tube growth. Important results in the biochemical and physiological aspects of molecular regulation have been reported. The following review attempts to cover these aspects. It is primarily based on talks presented by the authors at the 13th International Congress on Sexual Plant Reproduction and is mainly addressed to non-experts in the fields of electrophysiology and ion signalling. We aim to present a general overview of electrical currents, ion dynamics, and ion transporters in pollen, and their possible role during pollen tube germination and growth. Together with results on other tip-growing cells, a general model of pollen tube germination and growth as a self-organizing process is proposed.     

Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof-of-concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium-mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5-ER, under the control of the pollen-specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non-transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP-tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP-tagged pollen was easily distinguishable from non-transgenic pollen using an epifluorescence microscope.     

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes

We recently reported the involvement of the ubiquitin pathway in microgametophyte development, and a direct role for the 26S proteasome in regulating pollen tube emergence in kiwifruit. Here we show that the ubiquitin/proteasome proteolytic pathway is involved not only in early kiwifruit pollen tube organisation, but also in maintaining polarised growth of tubes. By immunofluorescence analysis we show that ubiquitin and ubiquitin-protein conjugates are distributed mainly at the apex of emerging tubes, in both untreated pollen grains and pollen grains treated with MG132, an inhibitor of proteasome function. In the latter case, polysiphonous germination occurred and all the emerging areas were highly fluorescent. By adding MG132 to pollen when normal tube growth had already been established, accumulation of ubiquitin-protein conjugates, as well as a drastic reduction in tube growth and dramatic modifications of tube tip morphology were observed. Significantly, differential interference contrast microscopy analysis demonstrated that the clear zone was largely reduced or absent, and the nuclei were disconnected in their movements, reaching, in some cases, the extreme apex of the tip. These findings provide evidence that the ubiquitin- and proteasome-dependent proteolytic system could modulate the abundance and/or activity of key regulatory proteins involved in pollen tube emergence and polarised growth.     

Fast loading ester fluorescent Ca<Superscript>2+</Superscript> and pH indicators into pollen of <Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>

Loading of Ca²⁺-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca²⁺ ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca²⁺ indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca²⁺ distribution increased in the three apertures of pollen after hydration and showed a Ca²⁺ gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca²⁺ activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH_i regulated pollen tube growth.