A cascade signal pathway occurs in self-incompatibility of Pyrus pyrifolia

Pear (Pyrus pyrifolia L.) possesses an S-RNase-based gametophytic self-incompatibility (GSI) system and S-RNase, the self-incompatibility (SI) determinant in the pistil, has also been implicated in the rejection of self-pollen and genetically identical pollen. We have demonstrated that S-RNase depolymerises actin cytoskeleton, triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube, which indicates programmed cell death (PCD) may occur in SI response of Pyrus pyrifolia. Recently, we have identified that S-RNase specifically disrupted tip-localized reactive oxygen species (ROS) of incompatible pollen tube via arrest of ROS formation in mitochondria and cell walls in Pyrus pyrifolia. Furthermore, tip-localized ROS disruption not only decreased the Ca²⁺ current and depolymerised the actin cytoskeleton, but it also induced nuclear DNA degradation in the pollen tube. The results mentioned above indicate that a cascade signal pathway may occur in SI of Pyrus pyrifolia and PCD is used to terminate the incompatible pollen tubes growth. In this addendum, we review the cascade signal pathway of Pyrus pyrifolia SI.Key words: S-RNase, programmed cell death, reactive oxygen species, actin cytoskeleton, Ca²⁺ current, nuclear DNA     

G蛋白调节剂和花柱S-核糖核酸酶对花粉管生长及其钙离子浓度的影响

以‘丰水’和‘幸水’梨花柱及花粉为试材,用激光共聚焦显微技术,研究了离体条件下G蛋白活性调节剂和花柱S-RNA酶对花粉管生长及其游离Ca~(2 )浓度的影响。结果表明:G蛋白激活剂CTX可促进花粉管生长,且可解除花柱S-RNA酶对自身花粉管生长的抑制作用;G蛋白抑制荆PTX和花柱S-RNA酶共同处理使异体的花粉管生长受到抑制。CTX处理使花粉管尖端区的[Ca~(2 )]_i明显升高,花柱S-RNA酶处理引起自身花粉管尖端区的[Ca~(2 )]_i梯度消失;CTX和花柱S-RNA酶共同处理则使自身花粉管内的[Ca~(2 )J_i表现出两者单独处理时的综合特征;而花柱S-RNA酶和PTX共同处理后,异体的花粉管内[Ca~(2 )]_i表现出先升高后下降的趋势。     

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.     

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.     

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.     

Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube

The actin cytoskeleton plays a crucial role in the growth and polarity of the pollen tube. Due to inconsistencies in the conventional preservation methods, we lack a unified view of the organization of actin microfilaments, especially in the apical domain, where tip growth occurs. In an attempt to improve fixation methods, we have developed a rapid freeze-whole mount procedure, in which growing pollen tubes (primarily lily) are frozen in liquid propane at –180°C, substituted at –80°C in acetone containing glutaraldehyde, rehydrated, quenched with sodium borohydride, and probed with antibodies. Confocal microscopy reveals a distinct organization of actin in the apical domain that consists of a dense cortical fringe or collar of microfilaments starting about 1–5 m behind the extreme apex and extending basally for an additional 5–10 m. In the shank of the pollen tube, basal to the fringe, actin forms abundant longitudinal filaments that are evenly dispersed throughout the cytoplasm. We have also developed an improved ambient-temperature chemical fixation procedure, modified from a protocol based on simultaneous fixation and phalloidin staining. We removed EGTA, elevated the pH to 9, and augmented the fixative with ethylene glycol bis[sulfosuccinimidylsuccinate] (sulfo-EGS). Notably, this protocol preserves the actin cytoskeleton in a pattern similar to that produced by cryofixation. These procedures provide a reproducible way to preserve the actin cytoskeleton; employing them, we find that a cortical fringe in the apex and finely dispersed longitudinal filaments in the shank are consistent features of the actin cytoskeleton.     

Cytochalasin B Treatment of Apple (<Emphasis Type="Italic">Malus pumila</Emphasis> Mill.) Pollen Tubes Alters the Cytoplasmic Calcium Gradient and Causes Major Changes in the Cell Wall Components

It is well established that the actin cytoskeleton is absolutely essential to pollen germination and tube growth. In this study we investigated the effects of cytochalasin B (CB), which affects actin polymerization by binding to the barbed end of actin filaments, on apple (Malus pumila Mill.) pollen tube growth. Results showed that CB altered the morphology of pollen tubes, which had a larger diameter than control tubes beside inhibiting pollen germination and tube growth. Meantime CB also caused an abnormal distribution of actin filaments in the shank of the treated pollen tubes. Fluo-3/AM labeling indicated that the gradient of cytosolic calcium ([Ca²⁺]c) in the pollen tube tip was abolished by exposure to CB, which induced a much stronger signal in the cytoplasm. Cellulose and callose distribution in the tube apex changed due to the CB treatment. Immunolabeling with different pectin and arabinogalactan protein (AGP) antibodies illustrated that CB induced an accumulation of pectins and AGPs in the tube cytoplasm and apex wall. The above results were further supported by Fourier-transform infrared (FTIR) analysis. The results suggest the disruption of actin can result in abnormal growth by disturbing the [Ca²⁺]c gradient and the distribution of cell wall components at the pollen tube apex.     

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     

用非固定的荧光探针方法观察紫萼花粉管肌动蛋白纤丝的形式

用非固定的、二甲基亚砜作为渗透剂的、异硫氰四甲基罗丹明标记的鬼笔环肽染色方法,观察了紫萼(Hosta caerulea Tratt.)未萌发的花粉粒及不同生长时期的花粉管中的肌动蛋白纤丝的形式。显示未萌发的花粉粒中具有结晶状的梭状体,为肌动蛋白的一种贮藏形式。花粉萌发时,这种梭状体转移到短的花粉管中,逐渐松解、分支和形成肌动蛋白纤丝交错的网络。在花粉管迅速生长和达到一定长度时,肌动蛋白纤丝形成以与花粉管长轴平行的细丝占优势的网络系统,这是在大多数情况中紫萼花粉管典型的肌动蛋白纤丝的形式。在某些条件下,在花粉管接近顶端的前部,肌动蛋白纤丝可集合成长的粗束,这种粗束也常有分支和并合。肌动强白纤丝一直分布到花粉管的末端。讨论了研究肌动蛋白纤丝的非固定方法的重要性和进一步研究花粉管肌动蛋白纤丝值得注意的问题。     

Pollen tube growth: coping with mechanical obstacles involves the cytoskeleton

Cellular growth and movement require both the control of direction and the physical capacity to generate forces. In animal cells directional control and growth forces are generated by the polymerization of and traction between the elements of the cytoskeleton. Whether actual forces generated by the cytoskeleton play a role in plant cell growth is largely unknown as the interplay between turgor and cell wall is considered to be the predominant structural feature in plant cell morphogenesis. We investigated the mechano-structural role of the cytoskeleton in the invasive growth of pollen tubes. These cells elongate rapidly by tip growth and have the ability to penetrate the stigmatic and stylar tissues in order to drill their way to the ovule. We used agents interfering with cytoskeletal functioning, latrunculin B and oryzalin, in combination with mechanical in vitro assays. While microtubule degradation had no significant effect on the pollen tubes’ capacity to invade a mechanical obstacle, latrunculin B decreased the pollen tubes’ ability to elongate in stiffened growth medium and to penetrate an obstacle. On the other hand, the ability to maintain a certain growth direction in vitro was affected by the degradation of microtubules but not actin filaments. To find out whether both cytoskeletal elements share functions or interact we used both drugs in combination resulting in a dramatic synergistic response. Fluorescent labeling revealed that the integrity of the microtubule cytoskeleton depends on the presence of actin filaments. In contrast, actin filaments seemed independent of the configuration of microtubules.     

Imaging the actin cytoskeleton in growing pollen tubes

Given the importance of the actin cytoskeleton to pollen tube growth, we have attempted to decipher its structure, organization and dynamic changes in living, growing pollen tubes of Nicotiana tabacum and Lilium formosanum, using three different GFP-labeled actin-binding domains. Because the intricate structure of the actin cytoskeleton in rapidly frozen pollen tubes was recently resolved, we now have a clear standard against which to compare the quality of labeling produced by these GFP-labeled probes. While GFP-talin, GFP-ADF and GFP-fimbrin show various aspects of the actin cytoskeleton structure, each marker produces a characteristic pattern of labeling, and none reveals the entire spectrum of actin. Whereas GFP-ADF, and to a lesser extent GFP-talin, label the fringe of actin in the apex, no similar structure is observed with GFP-fimbrin. Further, GFP-ADF only occasionally labels actin cables in the shank of the pollen tube, whereas GFP-fimbrin labels an abundance of fine filaments in this region, and GFP-talin bundles actin into a central cable in the core of the pollen tube surrounded by a few finer elements. High levels of expression of GFP-talin and GFP-fimbrin frequently cause structural rearrangements of the actin cytoskeleton of pollen tubes, and inhibit tip growth in a dose dependent manner. Most notably, GFP-talin results in thick cortical hoops of actin, transverse to the axis of growth, and GFP-fimbrin causes actin filaments to aggregate. Aberrations are seldom seen in pollen tubes expressing GFP-ADF. Although these markers are valuable tools to study the structure of the actin cytoskeleton of growing pollen tubes, given their ability to cause aberrations and to block pollen tube growth, we urge caution in their use. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Financial Source: National Science Foundation grant Nos. MCB-0077599 and MCB-0516852 to PKH EU Research Training Network TIPNET (project HPRN-CT-2002-00265), Brussels, Belgium, to BV     

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。     

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.     

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。     

Microtubules and microfilaments are both responsible for pollen tube elongation in the coniferPicea abies (Norway spruce)

Summary InPicea abies (Norway spruce), microtubules and actin microfllaments both form a dense matrix throughout the tube mainly parallel to the direction of elongation. In these conifer pollen tubes the organization of this matrix is different from that in angiosperms. This study tests our hypothesis that differences in cytoskeletal organization are responsible for differences in tube growth and physiology. Pollen grains were germinated in media containing cytoskeletal disrupters and analyzed for germination, tube length, tube branching, and tip swelling. Disruption of microtubules significantly inhibits tube elongation and induces tube branching and tip swelling. Tip swelling is probably caused by disruption of the microtubules in the tip that are perpendicular to the direction of elongation. Confocal microscopy indicates that colchicine and propyzamide cause fragmentation of microtubules throughout the tube. Oryzalin and amiprophosmethyl cause a complete loss of microtubules from the tip back toward the tube midpoint but leave microtubules intact from the midpoint back to the grain. Disruption of microfilaments by cytochalasins B and D and inhibition of myosin by N-ethylmaleimide or 2,3-butanedione monoxime stops tube growth and inhibits germination. Microfilament disruption induces short branches in tubes, probably originating from defective microfilament organization behind the tip. In addition, confocal microscopy coupled with microinjection of fluorescein-labeled phalloidin into actively growing pollen tubes indicates that microfllament bundles extend into the plastid-free zone at the tip but are specifically excluded from the growing tip. We conclude that microtubules and microfilaments coordinate to drive tip extension in conifer pollen tubes in a model that differs from angiosperms.     

Arabidopsis LIM proteins PLIM2a and PLIM2b regulate actin configuration during pollen tube growth

The pollen tube grows rapidly, exclusively at its tip, to deliver its sperm for fertilization. The polarized tip growth of pollen tubes is dependent on the highly dynamic actin cytoskeleton. Plant LIM proteins (named after initials of containing proteins Lin11, Isl-1, and Mec-3) have been shown to regulate actin bundling in different cells, however, their roles in pollen tube growth have remained obscure. Here, we report the function of Arabidopsis LIM proteins PLIM2a and PLIM2b in pollen tube growth. The PLIM2a mutation resulted in short and swollen Arabidopsis pollen tube with defective actin bundles. The expression of the construct green fluorescent protein (GFP)-PLIM2b led to fluorescence of the actin bundles in germinating pollen and also the long actin bundles along the growing pollen tubes in Arabidopsis, but not of the short and sparse actin bundles that characterize the tip regions of the pollen tubes. There is a partially redundant function between PLIM2a and PLIM2b in the shank actin bundle organization during Arabidopsis pollen tube growth, as PLIM2b could rescue for the defective shank actin bundles in PLIM2a mutation pollen tubes. This report suggests critical roles of PLIM2a/PLIM2b in actin configuration during Arabidopsis pollen germination and tube growth.     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

Pollen-pistil interaction in maize: effects on genetic variation of pollen traits

Various factors (pollen diameter, in vitro germination and tube length, in vivo growth rate in selfed and nonselfed styles) which could possibly contribute to the competitive ability of pollen were investigated on 30 Zea mays L. inbred lines. The only factor with which pollen diameter was positively correlated was in vitro pollen-tube growth. Traits related to the early stages of growth (in vitro germination, in vitro tube length, early in vivo pollen growth rate) were all positively correlated with each other, and these early characteristics were negatively correlated with late in vivo tube growth rate, which is largely influenced by the stylar genotype.