Distribution of G-actin is related to root hair growth of wheat

BACKGROUND AND AIMS: Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth. METHODS: G-actin was labelled by fluorescein isothiocyanate-DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate-phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy. KEY RESULTS: Thick F-actin bundles did not extend into a region of approx. 5-10 microm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs. CONCLUSIONS: These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.     

Distribution of F-actin and Microtubules in Pollen and Pollen Tube of Lilium davidii

F-actin and microtubules are important components of pollen tube, which have very important function in cytoplasm streaming of pollen tube. The authors observed the distribution of Factin and microtubules in the pollen tube of Lilium davidii Duch. by immunofluorescence technique and confocol laser scanning microscopy, through which some new results were obtained. 1. Chemical fixation could preserve F-actin well in pollen tube, so the relation between F-actin and microtubules could be studied by the methods of chemical fixation and fluorescence labelling in pollen tube. 2. F-actin bundles were absent near the pollen tube tip, while microtubules were abundant and web formed in the pollen tube tip. The authors found that the terminal of microtubules was closely associated with the plasma membrane in the pollen tube tip. 3. Only a few F-actin bundles co-exist with the microtubules in the pollen tube of Lilium davidii. The results provided new evidence for the fimction and relationship between F-actin and microtubules in the pollen tube.     

Actin polymerization promotes the reversal of streaming in the apex of pollen tubes

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.     

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.     

Rop GTPase-dependent dynamics of tip-localized F-actin controls tip growth in pollen tubes

Tip-growing pollen tubes provide a useful model system to study polar growth. Although roles for tip-focused calcium gradient and tip-localized Rho-family GTPase in pollen tube growth is established, the existence and function of tip-localized F-actin have been controversial. Using the green fluorescent protein-tagged actin-binding domain of mouse talin, we found a dynamic form of tip-localized F-actin in tobacco pollen tubes, termed short actin bundles (SABs). The dynamics of SABs during polar growth in pollen tubes is regulated by Rop1At, a Rop GTPase belonging to the Rho family. When overexpressed, Rop1At transformed SAB into a network of fine filaments and induced a transverse actin band behind the tip, leading to depolarized growth. These changes were due to ectopic Rop1At localization to the apical region of the plasma membrane and were suppressed by guanine dissociation inhibitor overexpression, which removed ectopically localized Rop1At. Rop GTPase-activating protein (RopGAP1) overexpression, or Latrunculin B treatments, also recovered normal actin organization and tip growth in Rop1At-overexpressing tubes. Moreover, overexpression of RopGAP1 alone disrupted SABs and inhibited growth. Finally, SAB oscillates and appears at the tip before growth. Together, these results indicate that the dynamics of tip actin are essential for tip growth and provide the first direct evidence to link Rho GTPase to actin organization in controlling cell polarity and polar growth in plants.     

An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes

Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.     

Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, cytoskeleton, and cell wall components

The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner, while hardly similar effects were detected when cysteine-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanate-phalloidin and beta-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.     

Differential roles of microtubule and actin-myosin cytoskeleton in the growth of Pinus pollen tubes

The behavior and role of the microtubule (MT) and actin-myosin components of the cytoskeleton during pollen tube growth in two species of Pinus were studied using anti--tubulin, rhodamine-phalloidin, anti-myosin, and the appropriate inhibitors. Within germinated pollen tubes MTs were arranged obliquely or transversely, but in elongated tubes they were arranged along the tube's long axis. MTs were localized in the tube tip region, excluding the basal part. Altered growth was found in pollen tubes treated with colchicine; the tips of many pollen tubes incubated in the liquid medium were branched and/or rounded, and those in the agar medium were divided into many branches. Both the branching and the rounding were considered to be caused by the disturbance of polarizing growth of the tube due to MT disorganization with colchicine treatment. Actin filaments (F-actin) were found in the major parts of many pollen tubes along their long axis, excluding the tip region. In a few tubes, however, F-actin was distributed throughout the tube. The areas in the pollen tube containing F-actin were filled with abundant cytoplasmic granules, but the areas without F-actin had very few granules. The tube nucleus, which migrated from the grain area into the tube, was closely associated with F-actin. Germination of pollen grains treated with cytochalasin B was little affected, but further tube elongation was inhibited. Myosin was identified on cytoplasmic granules and to a lesser extent on the tube nucleus in the pollen tubes. Several granules were attached to the nuclear envelope. Tube growth was completely inhibited by N-ethylmaleimide treatment. In generative cells that were retained in the pollen grain, both MT and F-actin networks were observed. Myosin was localized on the cytoplasmic granules but not on the cell surface. In conclusion, it was shown that actin-myosin and MTs were present in gymnospermous Pinus pollen tubes and it is suggested that the former contributed to outgrowth of the tubes and the latter contributed to polarized growth. Several differences in the behavior of cytoskeletal elements in generative cells compared to angiosperms were revealed and are discussed.     

Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2+

Pollen tubes show active cytoplasmic streaming. We isolated organelles from pollen tubes and tested their ability to slide along actin bundles in characean cell models. Here, we show that sliding of organelles was ATP-dependent and that motility was lost after N-ethylmaleimide or heat treatment of organelles. On the other hand, cytoplasmic streaming in pollen tube was inhibited by either N-ethylmaleimide or heat treatment. These results strongly indicate that cytoplasmic streaming in pollen tubes is supported by the "actomyosin"-ATP system. The velocity of organelle movement along characean actin bundles was much higher than that of the native streaming in pollen tubes. We suggested that pollen tube "myosin" has a capacity to move at a velocity of the same order of magnitude as that of characean myosin. Moreover, the motility was high at Ca2+ concentrations lower than 0.18 microM (pCa 6.8) but was inhibited at concentration higher than 4.5 microM (pCa 5.4). In conclusion, cytoplasmic streaming in pollen tubes is suggested to be regulated by Ca2+ through "myosin" inactivation.     

Comparison of F-actin fluorescent labeling methods in pollen tubes of Lilium davidii

Fluorescence labeling of F-actin in pollen tubes by various methods has produced inconsistent results in the literature. Here, we report that EGTA, which was always used in fixative buffers in the past and thought to help cytoskeleton stabilization, can significantly affect F-actin distribution and lead to the formation of thick F-actin bundles at the tip of the pollen tube. We also found that vacuum-infiltration for the first 5 min during pollen tube fixation can better preserve normal cytoplasm structure and F-actin distribution. In contrast, m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) treatment before chemical fixation resulted in a shortening of the free zone of thick F-actin bundles in the pollen tube tip. Taken together, our results suggest that exclusion of EGTA and MBS from the fixative buffer, in combination with vacuum-infiltration in the first 5 min of fixation, can improve F-actin fluorescence labeling in pollen tubes of Lilium davidii.Li Wang and Yi-Min Liu are considered joint first authors     

Growth inhibition and recovery in freeze-substitutedLilium longiflorum pollen tubes: structural effects of caffeine

Summary In an attempt to correlate structural effects with the known dissipation of the tip-focused Ca²⁺ gradient caused by caffeine, we have examined the ultrastructure of caffeine-treated lily pollen tubes prepared by rapid freeze fixation and freeze substitution. We show that treatment with caffeine results in a rapid rearrangement of secretory vesicles at the pollen tube tip; the normal cone-shaped array of vesicles is rapidly dispersed. In addition, microfilament bundles appear in the tip region, where they had previously been excluded. Delocalized vesicle fusion continues in the presence of caffeine but tube extension ceases. Removal of caffeine from the growth medium initially causes tip swelling, delocalized vesicle fusion and presence of microfilaments well into the tip before normal structure and growth resume, concurrent with the previously reported return to a normal Ca²⁺ gradient.Abbreviations ER endoplasmic reticulum - MES 2-[N-morpholino] ethanesulfonic acid - MFs microfilaments     

Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes.

The distribution of intracellular free calcium ions ([Ca2+]i) was measured in pollen tubes of Lilium longiflorum using video imaging microscopy and the calcium sensitive indicators fura-2 and quin-2. The mean [Ca2+]i in growing pollen tubes measured with fura-2 shows a maximum of 1.7 to 2.6 microM in the tube tip and decreases almost exponentially to 60 to 100 nM at 100 microns behind the tip. Using quin-2, the maximum [Ca2+]i was also found in the tube tip but with a lower Ca2+ concentration, namely 1 microM. Addition of the calcium channel blocker La3+ caused a decrease of the [Ca2+]i maximum in the tube tip, indicating a heterogeneous distribution of Ca2+ channels along the plasma membrane of pollen tubes. The [Ca2+]i increased after addition of vanadate or compound 48/80. This suggests an involvement of a calmodulin-dependent Ca2+ pump in generation of the Ca2+ gradient in lily pollen tubes. The high [Ca2+]i found in the tube tip with fura-2 seems to indicate the real Ca2+ concentration and is probably responsible for vesicle fusion, fragmentation of actin filaments, and inhibition of cytoplasmic streaming.     

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.     

Identification and Characterization of Higher Plant Myosins Responsible for Cytoplasmic Streaming

in vitro using these myosins and of localization studies using antiserum raised against each heavy chain, we suggested that both myosins are molecular motors for generating the motive force for cytoplasmic streaming in higher plant cells. The 170-kDa myosin is expressed not only in somatic cells but also in germinating pollen. In contrast, the 175-kDa myosin is distributed only in somatic cells. In the tip region of growing pollen tubes, it has been demonstrated that a tip-focused Ca²⁺ gradient is indispensable for growth and tube orientation. Cytoplasmic streaming in this region has been shown to be inactivated by high concentrations of Ca²⁺. The motile activity in vitro of 170-kDa myosin is suppressed by low (μM) levels of Ca²⁺ through its CaM light chain, suggesting that this suppression is one of the mechanisms for inactivating cytoplasmic streaming near the tip region of pollen tubes. The motile activity in vitro of 175-kDa myosin is also inhibited by Ca²⁺ at concentrations higher than 10⁻⁶M. It has been revealed that the elevation of cytosolic Ca²⁺ concentrations causes the cessation of cytoplasmic streaming even in somatic cells. Therefore, Ca²⁺-sensitivity of the motile activity of myosin appears to be a general molecular basis for Ca²⁺-induced cessation of cytoplasmic streaming. Received 6 September 2000/ Accepted in revised form 7 October 2000     

Cytoskeleton in Pollen and Pollen Tubes of Ginkgo biloba L.

The distribution of F-actin and microtubules was investigated in pollen and pollen tubes of Ginkgo biloba L. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. A dense F-actin network was found in hydrated Ginkgo pollen. When Ginkgo pollen was germinating,F-actin mesh was found under the plasma membrane from which the pollen tube would emerge. After pollen germination, F-actin bundles were distributed axially in long pollen tubes of G. biloba. Thick F-actin bundles and network were found in the tip of the Ginkgo pollen tube, which is opposite to the results reported for the pollen tubes of some angiosperms and conifers. In addition, a few circular F-actin bundles were found in Ginkgo pollen tubes. Using immunofluorescence labeling, a dense microtubule network was found in hydrated Ginkgo pollen under confocal microscope. In the Ginkgo pollen tube, the microtubules were distributed along the longitudinal axis and extended to the tip. These results suggest that the cytoskeleton may have an essential role in the germination of Ginkgo pollen and tube growth.     

Rhodamine-phalloidin staining of F-actin in pollen after dimethylsulphoxide permeabilization

Summary Bundles of actin filaments were observed in in vitro cultured pollen of Lilium longiflorum and Nicotiana tabacum which had been permeabilized in a buffered medium containing 5% dimethylsulphoxide (DMSO), EGTA, rhodaminephalloidin for F-actin staining, and sucrose as an osmoticum. In imbibed pollen grains, especially those of lily, numerous short bundles and small foci of F-actin were clearly visible. In germinated pollen grains, fine bundles of F-actin could be seen to converge at the aperture of the pollen grain. Along the entire length of the pollen tube, including the extreme tip, a dense three-dimensional netaxial distribution of actin filaments was observed. The F-actin patterns visualized after permeabilization with DMSO are much finer and more detailed than those observed after conventional fixation with formaldehyde.     

Calcium entry into pollen tubes

Growing pollen tubes require calcium to maintain a tip-focused cytosolic gradient and as a constituent of the constantly expanding cell wall. Advances in cell and molecular biology as well as electrophysiology implicate several candidate channels and receptors in the flow of calcium into the cell. In this review we discuss the channels that have been identified and consider the role of the growing tip cell wall acting as a sink for calcium thus accounting for differences in oscillatory phase between influx measured on the outside of the cell and changes in tip concentration inside the cell. We also briefly draw attention to uptake mechanisms that restrict and shape the calcium signature in the growing pollen tube.     

百合花粉及花粉管内微丝和微管的分布

利用免疫荧光定位及荧光定位方法,结合共焦激光扫描显微镜,对百合（ＬｉｌｉｕｍｄａｖｉｄｉＤｕｃｈ．）花粉及花粉管内微丝及微管的分布进行了观察,得出了一些新的结果：１化学固定方法可以较好地保存花粉和花粉管内的微丝,从而可以在此条件下较好地进行微管和微丝的双标记,并进行两者相互关系的研究;２在距花粉管顶端１０～２０μｍ的范围内,用化学固定及ＴＲＩＴＣ鬼笔碱标记显示微丝的存在是很微弱的,基本上无法看到明显的微丝束,而同时用免疫荧光法标记却发现此部位微管很丰富,在花粉管顶端微管形成浓密的网状,而且其末端与花粉管顶端质膜相连;３在花粉管中,只有少数微丝与微管相互平行排列,而其中大多数微丝骨架与微管骨架并不存在共分布现象。为了解花粉管内微管和微丝的功能及相互关系提供了新的证据。     

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.     

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.