洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架

用DGD包埋去包埋方法,观察了洋葱花粉母细胞中胞间连丝和胞质通道内的胞质骨架分布。结果发现,在花粉母细胞的胞间连丝内有胞质骨加分布,这些骨架纤维集结成束,穿过胞间连丝。在胞质通道内也有胞质骨架分布,但与胸间连丝内的骨困分布有所不同,主要表现为两种形式;骨架纤维致密或稀少。研究讨论了胞质骨架在胞间连丝和胞质通道内的作用。     

Structures of the plant trophic tract: Plastid stromules and cell-wall plasmodesmata

Plastid stromules and cell-wall plasmodesmata are special plant-cell stsructures. They were discovered a century and a half apart: stromules at the beginning of the 21st century and plasmodesmata the end of the 19th. The former and latter are intra- and intercellular fragments, respectively, of endoplasmic reticulum, which is a network for photosynthesis distribution in the plant body. Methods and history of discovery, structural similarities and differences, and series of functional interpretations are discussed. The origins of both structures are connected with photosynthesis and photosynthate export. Their tubular structure and transport function are similar. The mobility of both is under control of the actomyosin cytoskeleton. The temperature regimes of formation and functioning also are the same. Photosynthesis is possible at 0°C and even lower. The structures of the exporting network—stromules and plasmodesmata—do not form below 10°C, and after 20°C the numbers of the former and latter significantly increase in relation to growth of cytoskeleton plasticity. The structural and functional continuity of stromules and plasmodesmata are postulated as the mobile trophic tract of vascular plants.     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

小麦胚乳细胞内,细胞间胞质骨架分布格局的超微结构观察

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）幼嫩胚乳为材料,经ＴｒｉｔｏｎＸ１００抽提、ＤＧＤ（ｄｉｅｔｈｙｌｅｎｅｇｌｙｃｏｌｄｉｓｔｅａｒａｔｅ）渗透、包埋,制备去包埋剂超薄切片,对细胞内、细胞间胞质骨架的分布格局与特征进行了电镜观察。由所获图像可见,胞质骨架呈主要由微管、微丝组成的三维网络结构;特别值得注意的是,有不少５～７ｎｍ的微丝在多处从网络表层向胞壁界面方向突出,并时而可见其横贯分界壁连接相邻骨架网络而将相邻细胞骨架联成一体。胚乳组织中微丝的跨胞分布以两种形式存在,直径达１００～２００ｎｍ微丝束的跨越和单个微丝的分散贯穿,看来这与该组织中开放态胞间通道与正常胞间连丝同时并存相吻合。初步讨论了微丝参与正常胞间连丝结构的可能性。     

Ultrastructural Observations on the Distribution of the Intra-and Intercellular Cytoskeleton of the Endosperm Cells in Triticum aestivum

By means of Triton X-l00 extraction and DGD (diethylene glycol distearate) embedment-free section method the distribution pattern and characteristics of intra- and intercellular cytoskeleton of endosperm cells of Triticum aestivum L. were studied with electron microscopy. Threedimensional architecture of the cytoskeleton could be recognized as a meshwork mainly composed of microtubules (MT) and microfilaments (MF). Attention was stressed on the interface of the adjoining cytoskeletal frameworks where an attractive phenomenon observed was that the MF extruding from the surface of the cytoskeleton often traversed the whole wall boundary and connected the neighbouring frameworks into an entity. In the endosperm tissue two types of transcellular MF distribution could be distinguished, the MF in bundles traversing the enlarged intercellular channels and the MF individually penetrating the wall boundary; that seemed to coordinate with the co-presence of normal and modified plasmodesmata in the same wall. The above observations demonstrated the intercellular cytoskeletal continuity within the symplast and confirmed that the MF was the main constituent of the traversing cytoplasmic strands, the possibility of MF being organized as a structural element of the normal plasmodesmata was also discussed.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection.

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport

BACKGROUND: Individual plant cells are encased in a cell wall. To enable cell-to-cell communication, plants have evolved channels, termed plasmodesmata, to span thick walls and interconnect the cytoplasm between adjacent cells. How macromolecules pass through these channels is now beginning to be understood. RESULTS: Using two green fluorescent protein (GFP) reporters and a non-invasive transfection system, we assayed for intercellular macromolecular traffic in leaf epidermal cells. Plasmodesmata were found in different states of dilation. We could distinguish two forms of protein movement across plasmodesmata, non-targeted and targeted. Although leaves have generally been considered closed to non-specific transport of macromolecules, we found that 23% of the cells had plasmodesmatal channels in a dilated state, allowing GFP that was not targeted to plasmodesmata to move into neighboring cells. GFP fusions that were targeted to the cytoskeleton or to the endoplasmic reticulum did not move between cells, whereas those that were localized to the cytoplasm or nucleus diffused to neighboring cells in a size-dependent manner. Superimposed upon this non-specific exchange, proteins that were targeted to the plasmodesmata could transit efficiently between 62% of transfected cells. CONCLUSIONS: A significant population of leaf cells contain plasmodesmata in a dilated state, allowing macromolecular transport between cells. Protein movement potential is regulated by subcellular address and size. These parameters of protein movement illustrate how gradients of signaling macromolecules could be formed and regulated, and suggest that non-cell-autonomous development in plants may be more significant than previously assumed.     

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.     

The role of the ER and cytoskeleton in plant viral trafficking

Plant viruses spread from cell to cell via plasmodesmata, which bridge the rigid cell wall and connect adjacent cells. The spread of infection is aided by interactions between the virus and the host components. These interactions have been intensively studied to understand the crosstalk between pathogen and host. The use of green fluorescent protein has shed new light on the close association between viral movement proteins and elements of the cytoskeleton and the endomembrane system.     

在三维结构上对百合花粉母细胞actin的免疫定位

传统的切片仅仅能够显示样品的平面结构,不能用于细胞中三维网络结构的研究。笔者在DGD(diethylene glycol distearate)包埋去包埋的基础上,结合电镜免疫胶体金技术对大卫百合花粉母细胞胞间及胞内细胞的骨架系统进行了研究,观察到高反差细胞微梁结构的三维网络,actin这一细胞骨架的主要成员被定位在该微梁结构纤维上。三维结构上的研究表明,actin不但是植物细胞核及细胞质骨架的成员,而且也存在于胞间连接结构(胞质桥和胞间连丝)中,推测它可能与细胞融合有关。实验结果同时表明,三维结构免疫胶体金技术对于细胞骨架和核基质的结构蛋白研究是行之有效的。     

Untersuchungen zur Phytophthora-Prognose

Plasmodesmata are cytoplasmic bridges in plants through which intercellular communication occurs. This involves the transport of ions, photoassimilates, growth hormones as well as protein‐nucleic acid complexes. Although these molecules are rather small (< 1 kDa) plant viruses succeed in using these intercellular highways to transport their genome. These viruses alter the plasmodesmata in some way to allow the transport of such large molecules. This review deals with how plant viruses manage this with the help of movement proteins and the cytoskeleton.     

Characterization of the unconventional myosin VIII in plant cells and its localization at the post-cytokinetic cell wall

Myosins are a large superfamily of motor proteins which, in association with actin, are involved in intra- cellular motile processes. In addition to the conventional myosins involved in muscle contractility, there is, in animal cells, a wide range of unconventional myosins implicated in membrane-associated processes, such as vesicle transport and membrane dynamics. In plant cells, however, very little is known about myosins. We have raised an antibody to the recombinant tail region of Arabidopsis thaliana myosin 1 (a class VIII myosin) and used it in immunofluorescence and EM studies on root cells from cress and maize. The plant myosin VIII is found to be concentrated at newly formed cross walls at the stage in which the phragmoplast cytoskeleton has depolymerized and the new cell plate is beginning to mature. These walls are rich in plasmodesmata and we show that they are the regions where the longitudinal actin cables appear to attach. Myosin VIII appears to be localized in these plasmodesmata and we suggest that this protein is involved in maturation of the cell plate and the re-establishment of cytoplasmic actin cables at sites of intercellular communication.     

去包埋剂制片术揭示小麦珠被组织中微丝的跨胞分布

以DGD(diethylene glycol distearate)包埋、去包埋剂制片法,结合常规电镜制备术,观察研究了小麦珠被组织中胞间连丝的结构与分布特征及其与微丝的关系。试验结果表明,同层珠被细胞间壁上胞间连丝的分布密度比上下层珠被间的要显著地高得多;珠被中胞间连丝保持固有的正常结构,孔径30—40 nm,变动幅度不大,观察不到如作者以前在小麦珠心和胚乳中所见的那种胞间连丝经结构修饰而扩大、转变为开放态的景象。对去包埋剂制片的观察进一步揭示,从胞质骨架网络表层有纤细微丝以分散状向外伸展突入分界壁,时而可见各个微丝横贯分界壁而连接相邻骨架网络的图象。联系珠被中正常胞间连丝结构的稳定性与各个微丝以分散状跨越分界壁,讨论了微丝参与胞间连丝结构的可能性与可能模式。     

Cell-to-cell movement of three genera (+) ss RNA plant viruses

The current investigations of three genera plant virus cell-to-cell movement were presented. Viruses reveal different local transport strategies, but all of them are the results of virus factors–host components interactions. The Tobacco mosaic virus (TMV) does not require capsid protein for translocation through plasmodesmata but 30 K movement protein participates in this process. It was found direct or indirect TMV movement proteins host partners in Tobamovirus movement like: pectin methylesterase, movement protein binding 2C, chaperones or cytoskeleton components and endoplasmatic reticulum membranes. The Potex- and Potyvirus cell-to-cell movement is closely related to replication network. The PVX capsid protein and triple gene block protein system are responsible for efficient local transport. Potyviruses move through the plasmodesmata by involving viral encoded proteins but not specific movement proteins. While the Potyvirus is the biggest known plant virus genus, host components participating in or regulating directly its plasmodesmata-movement are still not clear.     

Observations on structure and differentiation in plasmodesmata

Summary The fine structure of plasmodesmata in a number of plant tissues has been examined following fixation in glutaraldehyde. The structure of plasmodesmata is not constant. Variations occur between species, tissues and between different cellular situations in a single tissue. The nature of these variations is described and related to current theories of the formation and function of plasmodesmata. Evidence is presented that the young cell wall after division contains a large number of plasmodesmata, probably functionally and structurally identical, and that the development process involves characteristic modifications both to the distribution of plasmodesmata within the wall, and to the structure of individual plasmodesmata. The probable importance of the endoplasmic reticulum is stressed in relation to the formation and functioning of plasmodesmata.     

The distribution of plasmodesmata and its relationship to morphogenesis in fern gametophytes

Fern (Onoclea sensibilis) gametophytes when grown in the dark form a linear file of cells (one-dimensional) called a protonema. In the light two-dimensional growth occurs which results in a heart-shaped prothallus one cell thick. The objective of this paper is to relate the most common pattern of cell division observed in developing gametophytes to the formation of the plasmodesmatal network. Since the prothalli are only two dimensional, we can easily determine from thin sections the total number and the density (number per unit surface area) of plasmodesmata at each developmental stage. As the prothallus grows the number of plasmodesmata increases 50-fold in the apical or meristematic cell. This number eventually reaches a plateau even though the density continues to increase with each new cell division. What is particularly striking is that both the number and density of plasmodesmata between adjacent cells is precisely determined. Furthermore, the pattern of plasmodesmata distribution is predictable so that (1) we can identify the apical meristematic cells by their plasmodesmata number, or density, as well as by their size, shape and location, (2) we can predict, again from plasmodesmata number, the location of a future wall of the apical cell prior to its actual formation, (3) we can show that the density of plasmodesmata in the triangular apical cell of the prothallus (14 plasmodesmata microns-2) is comparable to those reported for secretory glands which are known to have high rates of plasmodesmatal transport and (4) we can show that once the plasmodesmata have been formed during division, no subsequent change in the number of plasmodesmata occurs following cell plate formation.     

Targeting of TMV movement protein to plasmodesmata requires the actin/ER network: evidence from FRAP

Fluorescence recovery after photobleaching (FRAP) was used to study the mechanism by which fluorescent-protein-tagged movement protein (MP) of tobacco mosaic virus (TMV) is targeted to plasmodesmata (PD). The data show that fluorescence recovery in PD at the leading edge of an infection requires elements of the cortical actin/endoplasmic reticulum (ER) network and can occur in the absence of an intact microtubule (MT) cytoskeleton. Inhibitors of the actin cytoskeleton (latrunculin and cytochalasin) significantly inhibited MP targeting, while MT inhibitors (colchicine and oryzalin) did not. Application of sodium azide to infected cells implicated an active component of MP transfer to PD. Treatment of cells with Brefeldin A (BFA) at a concentration that caused reabsorption of the Golgi bodies into the ER (precluding secretion of viral MP) had no effect on MP targeting, while disruption of the cortical ER with higher concentrations of BFA caused significant inhibition. Our results support a model of TMV MP function in which targeting of MP to PD during infection is mediated by the actin/ER network.     

The cytoskeleton facilitates a three-dimensional symplasmic continuum in the long-lived ray and axial parenchyma cells of angiosperm trees

The microtubule (MT), microfilament (MF) and myosin components of the cytoskeleton were studied in the long-lived ray and axial parenchyma cells of the secondary xylem (wood) and secondary phloem of two angiosperm trees, Aesculus hippocastanum L. (horse-chestnut) and Populus tremula L. x P. tremuloides Michx. (hybrid aspen), using indirect immunofluorescence localisation and transmission electron microscopy. MTs and MFs were bundled and oriented axially (parallel to the cell's long axis) within all parenchyma cell types after they had fully differentiated. Additionally, actin and myosin were immunolocalised at the thin-walled membranes of the pits, which linked cells in neighbouring files of both ray and axial parenchyma, and at the pits between axial and ray parenchyma cells themselves. Anti-callose antibody immunolocated the plasmodesmata at the pit membranes, and in the same pattern as that of anti-myosin. Ray cells are important symplasmic pathways between the xylem and the phloem throughout the life of trees. We hypothesise that the MT and MF components of the cytoskeleton in the ray and axial parenchyma cells are involved in the transport of materials within those cells, and, in association with the acto-myosin of plasmodesmata at pit fields, are also important in intercellular transport. Thus, the symplasmic coupling between ray cells, between axial parenchyma cells, and between axial parenchyma and ray cells represents an extensive three-dimensional communication pathway permeating the tree from the phloem through the cambium into the wood. We suggest that this cytoskeletal pathway has an important role in delivery of photosynthate, and mobilised reserves, to the actively dividing cambium, and in the movement of materials to sites of reserve deposition, principally within the wood. This pathway could also have an important role in co-ordinating developmental processes throughout the tree.     

Actin-regulated water permeability of two transport channels of plasmodesmata in roots of winter wheat cultivars varying in drought resistance

In roots of 5-6-day old seedlings of three cultivars of the winter wheat, varying in drought-resistance: Bezostaya 1 (low resistant), Mironovskaya 808 (resistant), and Albidum 114 (highly resistant) water permeability of two transport channels of plasmodesmata was studied at the action of cytochalasin B, which is known to inhibit polymerization of cytoskeleton actin filaments, by a pulse method of NMR, on the background of increasing water loss in the seedlings. It has been found that the registered coefficients of water self diffusion, two of which (D2 and D3) depend on the water permeability of different transport channels of plasmodesmata, differ in opposite directions. This may suggest that in roots of drought-resistant plants, after a moderate water loss, a diffusive water flow through the cytoplasmic symplast increases (demonstrated by an increase of D2), while that through the vacuolar symplast decreases (seen by an increase of D3). After a high water loss in seedlings, we noticed an even greater increase in water permeability of the cytoplasmic symplast, and a decrease in water permeability of the vacuolar symplast, however, in the roots of low resistant cultivars these changes were poorly expressed, if at all. Under stress-less conditions cytochalasin B would result in an increased water transport through the cytoplasmic channel of plasmodesmata due apparently to a destruction of their actin-myosin sphincters. Both weak and average degrees of water loss would strengthen the cytochalasin B exerted influence on plasmodesmal water conductance, that may testify to a synergetic action of these two factors. After a significant water loss this action was kept only partially, because the inhibitor, on blocking the cytoplasmic channel, did increase at the same time the effect of water stress, limiting water flows through the vacuolar symplast and, simultaneously, raising the water inflow to the apoplast.