Dynamics of vacuoles and actin filaments in guard cells and their roles in stomatal movement

Vacuoles and actin filaments are important cytoarchitectures involved in guard cell function. The changes in the morphology and number of vacuoles and the regulation of ion channel activity in tonoplast of guard cells are essential for stomatal movement. A number of studies have investigated the regulation of ion channels in animal and plant cells; however, little is known about the regulating mechanism for vacuolar dynamics in stomatal movement. Actin filaments of guard cells are remodelling with the changes in the stomatal aperture; however, the dynamic functions of actin filaments in stomatal movement remain elusive. In this paper, we summarize the recent developments in the understanding of the dynamics of actin filaments and vacuoles of guard cells during stomatal movement. All relevant studies suggest that actin filaments might be involved in stomatal movement by regulating vacuolar dynamics and the ion channels in tonoplast. The future study could be focused on the linker protein mediating the interaction between actin filaments and tonoplast, which will provide insights into the interactive function of actin and vacuole in stomatal movement regulation.     

Array and distribution of actin filaments in guard cells contribute to the determination of stomatal aperture

Actin filaments in guard cells and their dynamics function in regulating stomatal movement. In this study, the array and distribution of actin filaments in guard cells during stomatal movement were studied with two vital labeling, microinjection of alexa-phalloidin in Vicia faba and expression of GFP-mTn in tobacco. We found that the random array of actin filaments in the most of the closed stomata changed to a ring-like array after stomatal open. And actin filaments, which were throughout the cytoplasm of guard cells of closed stomata (even distribution), were mainly found in the cortical cytoplasm in the case of open stomata (cortical distribution). These results revealed that the random array and even distribution of actin filaments in guard cells may be required for keeping the closed stomata; similarly, the ring-like array and cortical distribution of actin filaments function in sustaining open stomata. Furthermore, we found that actin depolymerization, the trait of moving stomata, facilitates the transformation of actin array and distribution with stomatal movement. So, the depolymerization of actin filaments was favorable for the changes of actin array and distribution in guard cells and thus facilitated stomatal movement.     

Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.     

保卫细胞中的微丝骨架

微丝骨架存在于多种植物的保卫细胞中,周质微丝骨架的排列和结构是动态的。越来越多的证据表明保卫细胞中的微丝骨架可作为信号调节物,对气孔的启闭运动起着重要的调控作用。本文综述了保卫细胞微丝骨架的标记方法、结构,以及其在气孔运动中的功能和作用机制的最新研究进展。     

保卫细胞中的微丝骨架

微丝骨架存在于多种植物的保卫细胞中,周质微丝骨架的排列和结构是动态的。越来越多的证据表明保卫细胞中的微丝骨架可作为信号调节物,对气孔的启闭运动起着重要的调控作用。本文综述了保卫细胞微丝骨架的标记方法、结构,以及其在气孔运动中的功能和作用机制的最新研究进展。     

Tubular actin filaments in tobacco guard cells

The dynamic remodeling of actin filaments in guard cells functions in stomatal movement regulation. In our previous study, we found that the stochastic dynamics of guard cell actin filaments play a role in chloroplast movement during stomatal movement. In our present study, we further found that tubular actin filaments were present in tobacco guard cells that express GFP-mouse talin; approximately 2.3 tubular structures per cell with a diameter and height in the range of 1–3 µm and 3–5 µm, respectively. Most of the tubular structures were found to be localized in the cytoplasm near the inner walls of the guard cells. Moreover, the tubular actin filaments altered their localization slowly in the guard cells of static stoma, but showed obvious remodeling, such as breakdown and re-formation, in moving guard cells. Tubular actin filaments were further found to be colocalized with the chloroplasts in guard cells, but their roles in stomatal movement regulation requires further investigation.Key words: actin dynamics, tubular actin filaments, chloroplast, guard cell, stomatal movementStomatal movement responses to surrounding environment are mediated by guard cell signaling.^1,2 Actin filaments within guard cells are dynamic cytoarchitectures and function in stomatal development and movement.³ Arrays of actin filaments in guard cells that are dependent on different stomatal apertures have also been reported in references ^4–7. For example, the random or longitudinal orientations of actin filaments in closed stomata change to a radial orientation or ring-like array after stomata opening.^5,6,8 The reorganization of the actin architecture during stomatal movement depends on the depolymerization and repolymerization of actin filaments in guard cells. In contrast to the traditional treadmill model of actin dynamic mechanisms, stochastic dynamics of actin have been revealed in plant cells, such as in the epidermal cells of hypocotyl and root, the pavement cells of Arabidopsis cotyledons, and the guard cells of tobacco (Nicotiana tabacum).^9–11 In this alternative system, the short actin fragments generated from severed long filaments can link with each other to form longer filaments by end-joining activity. The actin regulatory proteins, Arp2/3 complex, capping protein and actin depolymerizing factor (ADF)/cofilin, may also be involved in the stochastic dynamics of actin filaments.^12,13Using tobacco GFP-mouse talin expression lines, we have previously analyzed the stochastic dynamics of guard cell actin filaments and their roles in chloroplast displacement during stomatal movement.^6,11 We found from these analyses that another arrangement of actin filaments, i.e., tubular actin filaments, exists in the guard cells of these tobacco lines. We first found the circle-like actin filaments in 82% of the guard cells (counting 320 cells) in tobacco expressing GFPmouse talin when analyzing a single optical section (Fig. 1A). In a previous study of BY-2 cells expressing GFP-Lifeact labeled actin filaments, Smertenko et al. found similar structures, i.e., quoit-like structures or acquosomes in all of the plant tissues examined except growing root hairs.¹⁰ However, in our present analysis of serial sections, we determined that the circle-like actin filaments in the tobacco guard cells were long tubes (Fig. 1A), as the lengths (about 3–5 µm) of these structures were greater than their diameter (about 1–3 µm). Hence, we denoted these structures as tubular actin filaments to distinguish them from the circular conformations of actin filaments observed previously in other plant cell tissues.^10,14–19 About 2.3 of these tubular actin filaments were found per guard cell, which is less than the number of acquosomes reported in BY-2 cells (about 6.7 per cell).¹⁰ Analysis of serial optical sections at the z-axis revealed that the tubular actin filaments localize in the cytoplasm near the inner walls of the guard cells (Fig. 1B), which is similar to the distribution of chloroplasts in guard cells.¹¹ Longitudinal sections further revealed a colocalization of tubular actin filaments and chloroplasts (Fig. 1B).Open in a separate windowFigure 1Tubular actin filaments in the guard cells of a tobacco (Nicotiana tabacum) line expressing GFP-mouse talin. (A) Optical-sections (interval, 1.5 µm) of guard cells in a moving stoma showing tubular actin filaments (arrow heads). Frames (a1) and (a2) are cross sections of 1.5-µm-picture through the yellow and red lines, respectively, revealing the cross section of the circle structures are parallel lines (arrows). (B) Optical-sections of a stoma from the outer periclinal walls to the inner walls of the guard cells (interval, 1 µm). The tubular actin filaments (arrow heads) are localized in the cytoplasm near to the inner periclinal walls of guard cells. Frame (b1) is the guard cell on the right of the frame “4 µm”; (b2) is the cross section of b1 through the red line; and (b3) is a higher magnification image of the area encompassed by the white square in b2. Arrows indicate the colocalization between the tubular actin filaments and the chloroplast (indicated using a red pseudocolor). (C) Time-series imaging showing the movement of tubular actin filaments in the guard cells of static stomata. Frame (c1) comprises three images colored red (0 S), green (40 S) and blue (80 S), that are merged in a single frame to show the translocation of the tubular actin filaments (arrows). (D) Time-series images of the opening stomata showing the breakdown (arrows) and re-formation (arrowheads) of the tubular actin filaments. All images were captured using a Zeiss LSM 510 META confocal laser scanning microscope, as described by Wang et al.¹¹ Bars, 10 µm.We performed time-lapse imaging and found that the translocation of tubular actin filaments is slow in static stomata in which the distance between two tubular actin filaments typically increased from 2.22 to 2.50 µm after 80 sec (Fig. 1C). In moving stomata, however, the tubular actin filaments showed an obvious dynamic reorganization whereby they could be processed into short fragments and also reemerged after they had disintegrated (Fig. 1D). These results indicate that tubular actin filaments have stochastic dynamics that are similar to the long actin filaments of guard cells.¹¹ In our previous study, we found that the stochastic dynamics of actin filaments correlate with light-induced chloroplast movement in guard cells.¹¹ However, whether the dynamics of the tubular actin filaments are also involved in chloroplast movement during stomatal movement remains to be investigated. In cultured mesophyll cells which had been mechanically isolated from Zinnia elegans, Wilsen et al. previously found a close association between fully closed actin rings and chloroplasts.¹⁸ These authors further found that the average percentage of cells with free actin rings increased at the initial culture stage, and then decreased, which indicates that the formation of actin rings might be a response of the actin cytoskeleton to cellular stress or disturbance.¹⁸ The turgor pressure of guard cells is the fundamental basis of stomatal movement leading to changes in the shape, volume, wall structure, and membrane surface of guard cells.^20–24 We speculate from our current data that there is a relationship between tubular actin filaments and the shape changes of guard cells during stomatal movement.     

拟南芥微丝加帽蛋白β亚基参与壳梭孢素诱导的气孔开放过程

气孔是植物响应外源信号,与环境进行水分和气体交换的门户。由外源信号引起的保卫细胞微丝骨架动态变化在气孔运动中发挥重要作用,但是具体的精确调节机制仍不清楚。微丝结合蛋白家族(ABPs) 是微丝动态组装最直接的调控者,它们的作用不容忽视。本文运用反向遗传学,以微丝结合蛋白—加帽蛋白 (CP) β-亚基 (CPB) 突变体cpb-3为实验材料,探究其在壳梭孢素 (FC)诱导气孔开放中的作用。结果发现:离体叶片干燥3 h,cpb-3突变体的叶片失水率为63.45%,明显高于野生型的48.99%。气孔开度测量及激光共聚焦显微镜观察发现,cpb-3突变体的气孔开放程度以及微丝动态重排对FC分子更敏感。气孔开度相比野生型增大了20% (P<0.05),含辐射状微丝排布的保卫细胞数量比例增幅达到58.3%,比对照组高出18.5%。此外,非损伤微测技术记录保卫细胞Ca²⁺、K⁺等跨膜运输动态,FC处理下,cpb-3突变体保卫细胞中Ca²⁺外流速度升至212.86 pmol cm^-2s^-1,野生型仅为68.76 pmol cm^-2s^-1,明显快于野生型。且K⁺内流也有相同表现。综上表明,微丝加帽蛋白CP的β亚基CPB可能通过调节保卫细胞微丝骨架动态重排以及离子流动,在FC诱导的气孔运动中发挥重要的作用。     

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.     

Molecular and biochemical characterization of a novel actin bundling protein in Acanthamoeba

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 microM of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳。经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况。在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达。但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性。经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构。在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达。本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料。     

GFP-mTn融合蛋白对蓝猪耳生活细胞微丝骨架的标记

用农杆菌介导法将嵌合基因GFP-mTn(mTn是微丝结合蛋白Talin的微丝结合域,可以显示活体细胞中微丝的结构)导入蓝猪耳.经激光共聚焦显微镜观察了转基因植株的各种不同组织中融合蛋白的表达和分布情况.在叶片的表皮细胞、保卫细胞、根部的皮层细胞中有融合蛋白的不同程度表达.但仅在保卫细胞中微丝标记状况良好,显示基因表达的组织特异性.经光诱导处于开放态的气孔的保卫细胞微丝呈网状结构,在细胞内无规则分布;经黑暗诱导处于关闭态的气孔保卫细胞中微丝束沿保卫细胞纵轴排列,呈卷曲状分布,并观察到螺旋和环状的微丝结构.在转基因植株的其他部位,例如茎表皮细胞、根毛细胞和花粉粒中,未检测到目的基因的表达.本研究获得的转基因植株为研究气孔运动过程中微丝动态变化提供了有用的材料.     

Actin filaments regulate the adhesion between the plasma membrane and the cell wall of tobacco guard cells

During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM–CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW–CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.     

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments in the control of guard cell volume changes (M. Kim, P.K. Hepler, S.-O. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels in the guard cell play an important role in stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. Inward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current level, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels in plant cells.     

Abscisic acid-induced actin reorganization in guard cells of dayflower is mediated by cytosolic calcium levels and by protein kinase and protein phosphatase activities

In guard cells of open stomata under daylight, long actin filaments are arranged at the cortex, radiating out from the stomatal pore. Abscisic acid (ABA), a signal for stomatal closure, induces rapid depolymerization of cortical actin filaments and the slower formation of a new type of actin that is randomly oriented throughout the cell. This change in actin organization has been suggested to be important in signaling pathways involved in stomatal closing movement, since actin antagonists interfere with normal stomatal closing responses to ABA. Here we present evidence that the actin changes induced by ABA in guard cells of dayflower (Commelina communis) are mediated by cytosolic calcium levels and by protein phosphatase and protein kinase activities. Treatment of guard cells with CaCl2 induced changes in actin organization similar to those induced by ABA. Removal of extracellular calcium with EGTA inhibited ABA-induced actin changes. These results suggest that Ca2+ acts as a signal mediator in actin reorganization during guard cell response to ABA. A protein kinase inhibitor, staurosporine, inhibited actin reorganization in guard cells treated with ABA or CaCl2, and also increased the population of cells with long radial cortical actin filaments in untreated control cells. A protein phosphatase inhibitor, calyculin A, induced fragmentation of actin filaments in ABA- or CaCl2-treated cells and in control cells, and inhibited the formation of randomly oriented long actin filaments induced by ABA or CaCl2. These results suggest that protein kinase(s) and phosphatase(s) participate in actin remodeling in guard cells during ABA-induced stomatal closure.     

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491–1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin. Received: 1 October 1999 / Accepted: 29 November 1999     

Phototropin-dependent biased relocalization of cp-actin filaments can be induced even when chloroplast movement is inhibited

In a recent publication using an actin-visualized line of Arabidopsis (Ichikawa et al. 2011, ref. ¹¹), we reported a detailed analysis with higher time resolution on the dynamics of chloroplast actin filaments (cp-actin filaments) during chloroplast avoidance movement and demonstrated a good correlation between the biased configuration of cp-actin filaments and chloroplast movement. However, we could not conclusively determine whether the reorganization of cp-actin filaments into a biased configuration preceded actual chloroplast movement (and, thus, whether it could be a cause of the movement). In this report, we present clear evidence that the reorganization of cp-actin filaments into a biased distribution is induced even in the absence of the actual movement of chloroplasts. When the cells were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin ATPase, chloroplast motility was completely suppressed. Nevertheless, the disappearance and biased relocalization of cp-actin filaments toward the side of the prospective movement direction were induced by irradiation with a strong blue light microbeam. The results definitively indicate that the reorganization of cp-actin filaments is not an effect of chloroplast movement; however, it is feasible that the biased localization of cp-actin filaments is an event leading to chloroplast movement.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Extracellular calmodulin(CaM)plays significant roles in many physiological processes,but little is known about its mechanism of regulating stomatal movements.In this paper,whether CaM exists in the guard cell walls of Arabidopsis and whether depolymerization of actin cytoskeleton is involved in extracellular CaM-induced stomatal closing are investigated.It is found that CaM exists in guard cell walls of Arabidopsis,and its molecular weight is about 17 kD.Bioassay using CaM antagonists W7-agarose and anti-CaM serum shows that the endogenous extracellular CaM promotes stomatal closure and delays stomatal opening.The long radial actin filaments in guard cells undergo disruption in a time-dependent manner during exogenous CaM-induced stomatal closing.Pharmacological experiments show that depolymerization of actin cytoskeleton enhances the effect of exogenous CaM-induced stomatal closing and polymerization reduces the effect.We also find that exogenous CaM triggers an increase in [Ca2+]cyt of guard cells.If [Ca2+]cyt increase is blocked with EGTA,exogenous CaM-induced stomatal closure is inhibited.These results indicate that extracellular CaM causes elevation of [Ca2+]cyt in guard cells,subsequently resulting in disruption of actin filaments and finally leading to guard cells closure.     

Depolymerization of actin cytoskeleton is involved in stomatal closure-induced by extracellular calmodulin in Arabidopsis

CaM ubiquitously presents inside eukaryotic cells. CaM抯 gene expression and its subcellular localization are regulated by light, osmotic stress, pathogens, plant hormones, etc.[1]. Intracellular CaM of plant displays important functions in pathogenesis and wounding reaction[2] and hypersensitive response[3]. CaM has been found extracellular spaces in many plant species, such as soluble extracts of oat coleoptile cell walls[4], the wheat coleoptile cell walls[5], maize root tips cell walls[6…     

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.