The auxin response of actin is altered in the rice mutantYin-Yang

Summary The rice mutantYin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity inYin-Yang. However, in contrast to the wild type, cell elongation inYin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.Abbreviations CD cytochalasin D - EPC ethyl-N-phenylcarbamate     

A role for actin-driven secretion in auxin-induced growth

In epidermal cells of Zea mays coleoptiles, actin microfilaments are organized in fine strands during cell elongation, but are bundled in response to signals that inhibit growth. This bundling response is accompanied by an increased membrane association of extracted actin. Brefeldin A, an inhibitor of vesicle secretion, increases the membrane association of actin, causes a bundling of cortical actin microfilaments, and reduces the sensitivity of cell elongation to auxin. A model is proposed where auxin controls the dynamics of an actin subpopulation that guides vesicles loaded with components of the auxin-signaling machinery towards the cell poles.     

Response of actin microfilaments during phytochrome-controlled growth of maize seedlings

Summary In seedlings of maize (Zea mays L. cv. Percival), growth is controlled by the plant photoreceptor phytochrome. Whereas coleoptile growth is promoted by continuous far-red light, a dramatic block of mesocotyl elongation is observed. The response of the coleoptile is based entirely upon light-induced stimulation of cell elongation, whereas the response of the mesocotyl involves light-induced inhibition of cell elongation. The light response of actin microfilaments was followed over time in the epidermis by staining with fluorescence-labelled phalloidin. In contrast to the underlying tissue, epidermal cells are characterized by dense longitudinal bundles of microfilaments. These bundles become loosened during phases of rapid elongation (between 2–3 days in irradiated coleoptiles, between 5–6 days in dark-grown coleoptiles). The condensed bundles re-form when growth gradually ceases. The response of actin to light is fast. If etiolated mesocotyls are transferred to far-red light, condensation of microfilaments can be clearly seen 1 h after the onset of stimulation together with an almost complete block of mesocotyl elongation. The observations are discussed in relation to a possible role of actin microfilaments in the signal-dependent control of cell elongation.     

Probing the actin-auxin oscillator

The directional transport of the plant hormone auxin depends on transcellular gradients of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. To get insight into this question, actin bundling was induced by overexpression of the actin-binding domain of talin in tobacco BY-2 cells and in rice plants. This bundling can be reverted by addition of auxins, which allows to address the role of actin organization on the flux of auxin. In both systems, the reversion of a normal actin configuration can be restored by addition of exogenous auxins and this fully restores the respective auxin-dependent functions. These findings lead to a model of a self-referring regulatory circuit between polar auxin transport and actin organization. To further dissect the actin-auxin oscillator, we used photoactivated release of caged auxin in tobacco cells to demonstrate that auxin gradients can be manipulated at a subcellular level.Key words: actin, auxin, BY-2, caged compounds, cell division, coleoptile, rice, tobacco     

玉米胚芽鞘细胞伸长生长与富含羟脯氨酸蛋白质的关系

玉米胚芽鞘细胞伸长生长进程中,富含羟脯氨酸蛋白质的合成速率同细胞的伸长生长呈负相关,迅速伸长期较低,而伸长近终止阶段出现活性高峰。生长素促进的伸长生长与富含羟脯氨酸蛋白质的合成、累积相关。IAA使胚芽鞘高体切段细胞的伸长生长增加4倍多,细胞中较低的羟脯氨酸蛋白质的合成速率似有利于生长素的促进效应。生长素对伸长细胞中羟脯氨酸蛋白质的转运和在壁中累积有抑制作用。     

Buder revisited: cell and organ polarity during phototropism

The induction of a radial polarity by environmental stimuli was studied at the cellular and organ levels, with phototropism chosen as a model. The light gradient acting on the whole coleoptile was opposed to the light direction acting upon individual cells in the classical Buder experiment, irradiating from the inside out. Alternatively, the stimulus was administered to the coleoptile tip with a microbeam-irradiation device. Tropistic curvature was assayed as a marker for the response of the whole organ, whereas cell elongation and the orientation of cortical microtubules were taken as markers for the responses of individual cells. Upon tip irradiation, signals much faster than basipetal auxin transport migrate towards the base. The data are discussed in terms of an organ polarity that is the primary result of the asymmetric light signal and affects, in a second step, an endogenous radial polarity of epidermal cells.     

Physical strain-mediated microtubule reorientation in the epidermis of gravitropically or phototropically stimulated maize coleoptiles

During gravitropic and phototropic curvature of the maize coleoptile, the cortical microtubules (MTs) adjacent to the outer epidermal cell wall assume opposite orientations at the two sides of the organ. Starting from a uniformly random pattern during straight growth in darkness, the MTs reorientate perpendicularly to the organ axis at the outer (faster growing) side and parallel to the organ axis at the inner (slower growing) side. As similar reorientations can be induced during straight growth by increasing or decreasing the effective auxin concentration, it has been proposed that these reorientations may be used as a diagnostic test for assessing the auxin status of the epidermal cells during tropic curvature. This idea was tested by determining the MT orientations in the coleoptile of intact maize seedlings in which the gravitropic or phototropic curvature was prevented or inversed by an appropriate mechanical counterforce. Forces that just prevented the coleoptile from curving in a gravity or light field prevented reorientations of the MTs. Forces strong enough to overcompensate the tropic stimuli by enforcing curvature in the opposite direction induced reorientations of the MTs opposite to those produced by tropic stimulation. These results show that the MTs at the outer surface of the coleoptile respond to changes in mechanical tissue strain rather than to gravitropic or phototropic stimuli and associated changes at the level of auxin or any other element in the signal transduction chain between perception of tropic stimuli and asymmetric growth response. It is proposed that cortical MTs can act as strain gauges in a positive feed-back regulatory circle utilized for amplification and stabilization of environmentally induced changes in the direction of elongation growth.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

A cytochalasin-sensitive actin filament meshwork is a prerequisite for local wound wall deposition inNitella internodal cells

Summary Reorganization of the actin cytoskeleton following cell wall puncturing of characean internodal cells was studied by immunofluorescence and confocal laser scanning microscopy. Injury locally destroyed the parallel subcortical actin filament bundles and cortical actin strands that are characteristic of unwounded regions. At wounds, a delicate three-dimensional interlaced structure of actin strands, with meshes up to 5 m wide, formed by de novo assembly of isolated filaments and by the elongation of residual subcortical actin bundles and cortical actin strands. The actin meshwork persisted for up to 2 h, corresponding to the duration of intense wound wall secretion. Actin filament bundles continuous with the subcortical bundles outside the wound then regenerated, their parallel alignment probably assisted by endoplasmic flow. Cytochalasin D concentrations that arrested cytoplasmic streaming completely inhibited the formation of the actin meshwork, wound wall deposition and recovery of actin bundles. Concentrations that only reduced streaming velocity delayed meshwork formation and wound walls were thinner than in controls. The actual amount of F-actin within the meshwork, however, was clearly greater in the presence of low cytochalasin concentrations. In late stages of recovery, the actin bundles became very thick and intervening spaces became wider thereby forming a conspicuous, three-dimensional lattice that was continuous with interwebbing subcortical bundles and cortical actin around the periphery of the wound. Our experiments suggest that actin meshwork formation is a prerequisite for plasma membrane-directed transport of vesicles involved in wounding-induced exocytosis in characean internodes. Stabilization of the meshwork by subinhibitory concentrations of cytochalasin D is probably caused by actinbinding properties of the drug that either induce bundling or impede function of associated proteins.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - CLSM confocal laser scanning microscope (microscopy) - DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - MBS m-maleimidobenzoyl N-hydroxy-succinimide ester - PBS phosphate-buffered saline - SCAB subcortical actin bundle     

Polar auxin transport and auxin-induced elongation in the absence of cytoplasmic streaming

Summary When cytoplasmie streaming in oat and maize coleoptile cells is completely inhibited by cytochalasin B (CB), polar transport of auxin (indole-3-acetic acid) continues at a slightly reduced rate. Therefore, cytoplasmic streaming is not required for polar transport. Auxin induces elongation in CB-inhibited coleoptile and pea stem segments, but elongation rate is reduced about 40% by CB. Therefore, stimulation of cytoplasmic streaming cannot be the means by which auxin promotes cell elongation, but streaming may be beneficial to elongation growth although not essential to it. A more severe inhibition of elongation develops after several hours in CB. With coleoptiles this could be due to inhibition of sugar uptake; in pea tissue it may be due to permeability changes and cytoplasmic degeneration. CB does not disorganize or disorient microfilament bundles when it inhibits streaming in maize, but appears instead to cause hypercondensation of microfilament material.     

Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.     

Cortical Actin Filaments Potentially Interact with Cortical Microtubules in Regulating Polarity of Cell Expansion in Primary Roots of Maize (Zea mays L.)

Evidence is accumulating implicating cortical microtubules in the directional control of cell expansion. However, the role of actin filaments in this process is still uncertain. To determine the involvement of actin in cell elongation, the organization of actin filaments in primary roots of maize (Zea mays L.) was examined by use of an improved fluorochrome-conjugated phalloidin-labeling method. With this method, a previously undetected state of actin organization was revealed in the elongation and maturation zone of maize roots. Fine transversely oriented cortical actin was observed in all cells of the elongation zone, including the epidermis, cortex, and vascular tissues. The orientation of cortical actin shifted from a predominantly transverse orientation to oblique, longitudinal, and/or random arrangements as the cells matured. The reorientation of cortical actin in maturing root cells mimics the behavior of cortical microtubules reported in other studies. Furthermore, roots treated with the microtubule-stabilizing drug taxol improved the quality of actin preservation as evidenced by the thicker bundles of cortical actin. This suggested that taxol was also capable of stabilizing the cortical actin networks. The elongation of roots exposed to 1 micromole Latrunculin B, an actin-disrupting drug, was inhibited, and after 24 h the roots exhibited moderate swelling particularly along the elongation zone. Latrunculin B also caused microtubules to reorient from transverse to oblique arrays. The results from this study provide evidence that cortical microtubules and actin filaments respond in a coordinated way to environmental signals and may well depend on both elements of the cytoskeleton.     

Microinjection of pollen‐specific actin‐depolymerizing factor, ZmADF1, reorientates F‐actin strands in Tradescantia stamen hair cells

Maize actin-depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein-conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re-modelling the actin after ADF1 injection may be provided by the microtubular system.     

Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC

Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal-dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the antimicrotubular drug ethyl-N-phenylcarbamate (EPC). The mutation EPC-Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild-type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild-type, microtubules did not reorient in response to auxin, and their response to microtubule-eliminating and microtubule-stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose-response for callus induction did not show any significant differences between wild-type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin-triggered signal transduction and microtubule reorientation.     

绿色荧光蛋白基因结合鼠Talin基因蛋白标记转基因水稻未成熟花粉的微丝骨架

利用绿色荧光蛋白基因结合鼠Talin基因表达技术及水稻转基因技术,在未成熟花粉发育期（即生殖细胞在形成后从靠壁部位移向中央部位的阶段）的水稻（Oryza sativa L.)内发现了一系列前人未曾报道过的微丝骨架的形成和多变过程。在这一发育阶段,未成熟花粉内的生殖细胞呈圆形,中央部位存有一个大液泡,大量微丝在细胞的中央胞质内形成。微丝首先在营养核的核膜表面形成两个集结中心,中心内的微丝呈短粗状。尔后,中心微丝不断瞎长,最终在细胞中央的胞质内形成一个非常 类似多个纺锤体结合在一起的网络结构。这一网络的中间部位经常包围着营养核和生殖细胞,网络的部分微丝则与存在周缘细胞质（或称周质）的微丝网络形成连接,在连接点部位则形成一些由微丝环状组成的结构。未成熟花粉中央的微丝网络可能与营养核和生殖细胞在未成熟花粉内的运动有密切关系。     

Immunofluorescent visualization of arrays of transverse cortical actin microfilaments in wheat root-tip cells

Summary Actin microfilaments in isolated root-tip cells from wheat (Triticum aestivum L. cv. Kite) were visualized by immunofluorescence microscopy using two different antiactin monoclonal antibodies. Cells in interphase contain predominantly subcortical bundles of microfilaments, as described in many cell types, but in preprophase and prophase cells, immunodetectable actin is organized solely in ordered arrays of cortical microfilaments that cover the entire surface of the cell, transverse on lateral faces, random on end walls. Intermediate stages with random and transverse microfilaments are also seen on lateral faces. The cell cycle stage-dependent transverse cortical microfilaments described here are previously unreported in higher plant cells.Abbreviations Ig immunoglobulin - MF microfilament     

Some of eukaryotic elongation factor 2 is colocalized with actin microfilament bundles in mouse embryo fibroblasts

Indirect immunofluorescent microscopy was used to study the distribution of eukaryotic elongation factor 2 (EF-2) in cultured mouse embryo fibroblasts. The perinuclear area (endoplasm) of all the cells and many straight cables running along the whole cytoplasm were stained with monospecific goat or rabbit antibodies to rat liver EF-2. Double staining of the cells with antibodies to EF-2 and rhodaminyl-phalloidin (used for actin microfilament detection) showed that EF-2 containing cables coincided with bundles of actin microfilaments. Not all actin microfilament bundles contained EF-2: sometimes EF-2 was not observed in bundles running along the cell edges or in actin microfilament junctions. Triton X-100 extracted most of EF-2 from the cells and no actin microfilament bundles were stained with the EF-2 antibodies in the Triton-extracted cells. Thus, in mouse embryo fibroblasts EF-2 can be found along actin microfilament bundles, but it is unlikely to be their integral protein.     

The organization of the actin cytoskeleton in vertical and graviresponding primary roots of maize

To determine whether actin microfilament (MF) organization is correlated with differential elongation, primary roots of Zea mays cv Merit maintained vertically or reoriented horizontally for 15 to 120 min were stained with rhodamine phalloidin and examined with a confocal microscope. Root curvature was measured with a computer-controlled video digitizer. In vertical roots bundles of MFs in the elongation and maturation zone were oriented parallel to the longitudinal axis of cells. MFs in the vascular parenchyma cells were more abundant than in the cortex and epidermis. Epidermal and proendodermal cells in the meristematic region contained transverse cortical MFs. The organization of MFs of graviresponding roots was similar to that of vertical roots. Application of cytochalasin B or cytochalasin D resulted in extensive disruption of MFs in the cortex and epidermis, but only partially affected MFs in the stele. Despite the cytochalasin B-induced depolymerization of MFs, gravicurvature exceeded that of controls. In contrast, the auxin transport inhibitor N-1 naphthylphthalamic acid suppressed root curvature but had no observable effect on the integrity of the MFs. The data indicate that MFs may not be involved in the graviresponse of maize roots.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.